Pan-bacterial and pan-fungal identification reagents and methods of use thereof

ABSTRACT

Murein binding polypeptide and antibiotic diagnostic reagents, methods and kits for detecting eubacteria and fungus in biological samples.

This application is a division of U.S. patent application Ser. No.08/823,293 filed Mar. 21, 1997, now U.S. Pat. No. 5,935,804.

FIELD OF THE INVENTION

The invention generally relates to microbiology and reagents and methodsfor identifying fungi and bacteria in samples of biological fluids,foods, water, air, solutions and the like; and, specifically, todiagnostic reagents and methods for detecting and quantifying fungi andbacteria, as well as, for distinguishing between bacteria and fungi in asample.

BACKGROUND OF THE INVENTION

Rapid tests for identification of pathogenic bacteria, fungi and theirproducts are becoming increasingly important to health careprofessionals, as well as individuals responsible for water and foodsafety. Timely identification and generic classification of etiologicagents is a key to preventing spread of disease, and is also effectiveto expedite patient treatment and thereby reduce costs associated withdisease management.

Microbiological stains are diagnostic reagents capable of identifyingbacteria and fungi from cultures, but they commonly are not useful asrapid diagnostic tests with patient samples because of cross-reactivitywith natural mammalian and plant products and with other non-pathogenicmicroorganisms. While gram-staining for bacteria remains a most usefuldiagnostic criteria in evaluation of isolated bacterial cultures, thereagents and methods generally lacks sensitivity because many groups ofbacteria stain either poorly or not at all. Similarly, histologicalstaining of polysaccharides using Grocott methenamine silver nitrate asa test for fungi lacks sensitivity and is subject to confusingnon-specific cross-reactions with e.g. connective tissue proteoglycans,glycosaminoglycans and mucins. Commonly, definitive identification andconfirmation of infection, or contamination, requires multiple tedioussteps of culture and multiple different testing methods and often theseprocedures are not suitable for use in smaller clinical testlaboratories.

Bacterial envelopes are composed of an inner phospholipid bilayer withmembrane proteins surrounded by a rigid shell of covalently linkedpeptidoglycan that is reactive with crystal violet and iodine, i.e., theenvelope in gram positive bacteria. Gram-negative bacteria have thelatter structure, but with lesser amounts of peptidoglycan, and theirenvelope includes an additional outer membrane characterized bylipopolysaccharide (LPS), porins and transport proteins. Peptidoglycanstructures vary in different bacteria. In E. coli peptidoglycan,N-acetylglucosamine alternates with N-acetylmuramic acid inβ(1,4)-glycosidic bonds to form complex polymers with tetrapeptide sidechains. The latter side chains are composed ofL-Ala-D-Glu-mesodiaminopimelic acid-D-Ala. Cross-linking betweenpeptidoglycan chains forms a gel that varies in consistency dependentupon the degree of cross-linking.

In contrast, most pathogenic fungi contain chitin in their cell walls,septa and spores, both in hyphal and yeast forms. Chitin is a β1→4linked polymer of only 2-deoxy-2-acetamindoglucose (N-acetylglucosamine, abbreviated GlcNAc). Chitin is also found in tissues ofinsects and crustaceans. Certain classes of fungi have cell walls thatcontain both chitins and murein-like compounds. However, the murein-likecompounds are commonly hidden within thick protective cell wallstructures, and/or expressed only in low levels. Fungi, as eukaryotes,also have many similarities with mammalian cells, often making it moredifficult to distinguish between patient cellular materials and fungalproducts.

Immunoassays used in clinical microbiology commonly rely on antibodiesthat, while highly specific, are also narrowly reactive, e.g., with asingle defined epitope in a complex carbohydrate structure of aparticular serotype of bacteria. Cross-reactivity of reagents with morethan one different type of bacteria is often viewed as an undesirableperformance attribute. Antibody reagents also frequently are unable todistinguish between a whole bacteria and the degradative products of abacteria, and as a result breakdown products can act as `confounding`,or `interfering, substances` in diagnostic assays. Where enzymes areused in immunoassays, they are commonly used to generate a detectablesignal. For example, enzyme-linked immunosorbent assays (ELISA) such asthose disclosed in U.S. Pat. Nos. 4,233,402 and 4,486,530, involvelabeling an antibody (or antigen) by covalently linking it to acatalytically active enzyme. The presence (or amount) of the labeledcompound may be determined in an assay by adding an enzyme substratethat produces a measurable signal (e.g., a colored product orfluorescence.) While certain assay formats rely upon re-activation of aninactivated-enzyme (e.g., U.S. Pat. No. 4,043,872), generation of asignal in an ELISA commonly requires a catalytically active enzyme, andpreferably one having giving a rapid production of product (i.e., a highturnover number.) Catalytically active enzymes considered for possibleuse in diagnostic immunoassays include those hydrolyzing glycosidicbonds (e.g., U.S. Pat. Nos. 4,208,479 at column 17; 4,299,916 at column33.)

Enzymes are given names indicating both the principal substrate and thereaction catalyzed. However, few enzymes are absolutely specific to thestructure of a particular substrate and most can act on closely relatedstructural analogues of their physiological substrates, although usuallyat reduced rates. The Commission on Enzymes of the International Unionof Biochemistry has evolved a systematic nomenclature for enzymes basedon the reactions catalyzed. Lysozyme, classified as a glycosidehydrolase in IUB class EC3.2.1 (i.e., IUB class EC3.2.1.17), hasspecificity for compounds containing N-acetylmuramic acid and a peptideside chain (i.e., mureins.) Lysozymes from different species catalyzehydrolysis of β(1,4) bonds between N-acetylmuramic acid and adjacentsugar residues in mureins and chitins, but chitins more slowly.Chitinase, classified as an N-glycosyl hydrolase (i.e., IUB classEC3.2.1.14), binds and degrades chitin and murein, but murein much moreslowly than chitin.

It would be highly desirable for clinical test laboratories to haveaccess to reagents that, while specifically reactive with many genera ofbacteria and fungi, are also useful as reagents in assays thatdistinguish between bacterial and fungal infection or contamination.However, the array of different antigens in bacteria and fungi that areavailable as potential targets for development of immunoassays issomewhat bewildering. Also, increasingly laboratory personnel are beingplaced at a potential risk of exposure to debilitating or lifethreatening diseases by contact with infected patient samples. While itis commonly an aim to conduct all assays with non-infectious materials,the resultant fixed and killed bacterial samples often contain denaturedantigens that are poorly reactive with assay reagents. Diagnosticreagents reactive with fixed, and/or killed and dead bacterial andfungal products are highly desirable.

Bacteria, fungi and their products are often present in samples invanishingly small amounts. Recently, attempts have been made to developsensitive alternatives to immunoassays by using polymerase chainreaction (PCR) and enzyme-linked oligonucleotide probe methods.(InfectioDiagnostics Corporation of Quebec, Canada has disclosed a PCRtest method for detection of bacteria.) Unfortunately, these testmethods are often time-consuming, e.g. requiring repetitive thermalcycling for amplification, and also requiring highly trained personneland special laboratory conditions to insure optimal performance.Reagents and methods capable of rapidly detecting very small numbers ofbacteria and fungi in samples would be of great importance in clinicaltesting of patient materials, as well as in monitoring food, air andwater.

Objects of the invention provide reagents and assays for detecting smallnumbers of killed and treated bacteria and fungi from a wide range ofdifferent genera. The reagents and assays are unreactive with anyproducts in normal mammalian tissues and are also capable, in certainobjects, of distinguishing between bacterial and fungal infection orcontamination. Other objects provide a variety of methods for detectingeubacteria which do not require any special personnel training orfacilities for optimizing their use.

SUMMARY OF THE INVENTION

Disclosed herein are reagents and methods for identifying eubacteria,fungi and their products in a wide range of different biological tissuesand fluids including patient and veterinary samples, food, air andwater. The diagnostic reagents of the invention contain non-antibodymurein-binding polypeptides that are substantially purified andpreferably modified from their native structure or amino acid sequence,e.g. through genetic or chemical modification(s), to render them mosteffective in the disclosed methods. Murein-binding polypeptides haveamino acid sequences capable of binding to denatured bacterial mureinligands and fungal murein-like ligands as they appear in fixed anddenatured bacterial and fungal particles. Murein-binding polypeptidesare reactive with the latter murein ligands as they appear in alcoholfixed (e.g., 80% ethanol), or alkaline denatured (e.g., 2M NaOH, 60° C.for 30 minutes) and/or protease treated (e.g., 0.25% trypsin, 37° C., 30minutes), and/or periodate-treated, and/or acetic anhydride-treated deadbacterial and fungal particles. Binding of the murein bindingpolypeptides to the murein ligands is high affinity and specific withlittle or no detectable cross-reactivity with mammalian or plantproducts. Non-antibody murein-binding proteins include native andcatalytically disabled enzymes selected from among murein biosyntheticand hydrolytic enzymes produced by mammals, insects, bacteria,bacteriophage and fungi.

Murein-binding polypeptides are preferably purified substantially foruse and may be chemically conjugated to a signal generating compoundsuch as a fluorophore, a magnetic particle, a latex bead or an enzyme,and may also be linked to biotin or avidin. In-situ binding,enzyme-substrate (ISBES) assay methods, disclosed herein, employ themurein-binding polypeptide reagents to detect and quantify bacteria andfungi in-situ, i.e. without isolation and culture. The assay methodsinclude steps for detection of bacteria and fungi in fixed tissuesamples by histochemistry; in samples of bodily fluids such as urine byflow cytometry or by enzyme-linked solid phase methods; in blood bymachine assay methods or latex dipstick methods; and, in food, air,water samples by dot-blot methods. ISBES sample preparation methodsinclude the step of treating a biological sample to simultaneouslyeffect the following: namely, (i) killing infectious bacterial, fungalor viral agents in the sample to produce dead bacterial and fungalparticles; (ii) lysing mammalian cells such as erythrocytes andleukocytes; (iii) disaggregating bacterial and fungal rafts into uniformsingle cell/particle suspensions; (iv) dissociating bacterial and fungalparticles from other possible particulate materials in the samples suchas, plant cellulosic and lignin materials; (v) removing exterior fungalcell wall surface layers to expose murein-like compounds in the deadparticles; (vi) removing/denaturing mammalian cellular materials thatmight cross-react in the assay and contribute to nonspecific background;(vii) denaturing bacterial and fungal cell surface polypeptides toincrease susceptibility to protease treatments and/or to increaseaccessibility of murein-like ligands for binding to murein bindingpolypeptides; and, while (viii) retaining the ability of the deaddenatured bacterial and fungal particles to react with natural andmodified murein binding polypeptides.

Murein binding polypeptide reagents and ISBES assay methods offercost-savings and other commercial advantages (e.g., shelf lifestability) for manufacturers and users of diagnostic test reagents, kitsand reagent packages marketed for detecting bacterial and fungalinfections. In addition, quantitative and qualitative flow cytometry andcytofluorimetric ISBES methods are disclosed for determining thepresence, severity and type of microbial infection in a patient bydetecting and quantifying the number of bacterial and/or fungi in abiological sample which are reactive with a murein binding polypeptidereagent. The latter particle size and light scattering ISBES methodsallow rapid quantification and/or discrimination between bacteria andfungi in patient samples, and are not effected by the presence of cellwall fragments. Kits are also disclosed for identifying eubacteria andfungi in samples of air, water and biological samples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the background auto-fluorescence of bacteria as measuredby flow fluorimetry.

FIG. 1B shows binding of an F-NHS-conjugated murein-binding protein(MBP) to bacteria as visualized by cytofluorimetric analysis andplotting fluorescence intensity (horizontal axis) against the number ofincidents (i.e., particles counted; vertical axis) to form a histogramplot as disclosed further in EXAMPLE 5, below.

FIG. 2A shows a fluorescence photomicrograph of staining of clusters,rafts and aggregates of fungal cells in a urine sediment sample asvisualized by staining with an MBP-F-NHS-conjugate, as disclosed inEXAMPLE 8, below.

FIG. 2B shows a photomicrograph of fluorescence staining of killed anddenatured fungal particles after treatments and staining according tothe methods of the invention, as illustrated in EXAMPLE 8, below.

FIG. 2C shows a plot of fluorescence intensity (x axis;FSC-H/FSC-Height) against particle size (y axis: SSC-H/SSC-Height) forunstained negative control urine sediment residue particles in a normalhuman urine sample that was subject to chemical treatments andfluorescent staining with an MBP-conjugate according to the invention,as disclosed further below and illustrated in EXAMPLE 8. The circlelabeled "YG", yeast gate, denotes the data point region where killed anddenatured fungal particles, if present, would be recorded.

FIG. 2D shows a plot of fluorescence intensity (x axis) versus particlesize (y axis) for a negative control urine sediment sample prior to anychemical treatments; as disclosed further in EXAMPLE 8, below.

FIG. 2E shows a plot of fluorescence intensity (x axis) versus particlesize (y axis) for a biological sample containing fungal particles whichare indistinguishable in this analysis from normal human urine sedimentparticles; as disclosed further in EXAMPLE 8, below.

FIG. 2F shows a plot of fluorescence intensity (x axis) versus particlesize (y axis) for the fungal sample of FIG. 2E, above, after chemicaltreatments and fluorescence staining with an MBP-conjugate according tothe methods of the invention, and as disclosed below and illustrated inEXAMPLE 8. The circle labeled "YG" contains data points recordedprimarily from killed and denatured singlet and doublet fungal particlesand is separated from the majority of the other urine sedimentparticles.

FIG. 2G shows a plot of the number of fungal particles (y axis) againstfluorescence intensity (x axis) before chemically treating or staining anegative control biological sample, as disclosed further in EXAMPLE 8,below.

FIG. 2H shows a plot of the number of fungal particles (y axis) againstfluorescence intensity (x axis) before chemical treatment or staining anegative control fungal sample in buffer; as disclosed further inEXAMPLE 8, below.

FIG. 2I shows a plot of the number of killed and denatured fungalparticles (y axis) against fluorescence intensity (x axis) afteralkaline treatment and then staining a biological sample withMBP-conjugate, as disclosed further in EXAMPLE 8, below.

FIG. 2J shows a plot of the number of killed and denatured fungalparticles (y axis) against fluorescence intensity (x axis) afteralkaline treatment and then staining a positive control fungal sample inbuffer with MBP-conjugate, as disclosed further in EXAMPLE 8, below.

FIG. 2K shows a plot of the number of killed and denatured fungalparticles (y axis) against fluorescence intensity (x axis) afteralkaline- and acetylation-treatments, but before staining a firstpositive control fungal biological urine sample, as disclosed further inEXAMPLE 8, below.

FIG. 2L shows a plot of the number of killed and denatured fungalparticles (y axis) against fluorescence intensity (x axis) afteralkaline- and acetylation-treatments and staining a first positivecontrol fungal biological urine sample with MBP-conjugate, as disclosedfurther in EXAMPLE 8, below.

FIG. 2M shows a plot of the number of killed and denatured fungalparticles (y axis) against fluorescence intensity (x axis) afteralkaline- and acetylation-treatments, but before staining a secondpositive control fungal biological urine sample, as disclosed further inEXAMPLE 8, below.

FIG. 2N shows a plot of the number of killed and denatured fungalparticles (y axis) against fluorescence intensity (x axis) afteralkaline- and acetylation-treatments and staining a second positivecontrol fungal biological urine sample with MBP-conjugate, as disclosedfurther in EXAMPLE 8, below.

FIG. 3A depicts graphically the results of an experiment in whichdiffering volumes (μl) of a fungal cell suspension were added toaliquots of a normal human urine sediment sample to achieve cellconcentrations in the range of about 1,000-5,000 cells/ml urine. Theindividual aliquot samples were then prepared according to the alkaline-and protease-treatments and stained with MBP-conjugate according to themethods of the invention, as disclosed further below and illustrated inEXAMPLE 8.

FIG. 3B depicts graphically the results of an experiment in whichdiffering volumes (μl) of a fungal cell suspension were added toaliquots of a normal human urine sediment sample to achieve cellconcentrations in the range of about 5,000-20,000 cells/ml urine. Theindividual aliquot samples were then prepared according to the alkaline-and protease-treatments and stained with MBP-conjugate according to themethods of the invention, as disclosed further below and illustrated inEXAMPLE 8.

FIG. 4 depicts graphically the results of an experiment in whichdiffering volumes (μl) of a fungal cell suspension were added toaliquots of a normal human urine sediment sample to achieve cellconcentrations in the range of about 5,000-200,000 cells/ml urine. Theindividual aliquot samples were then prepared according to the alkaline-and protease-treatments and stained with MBP-conjugate according to themethods of the invention, as disclosed further below and illustrated inEXAMPLE 8.

FIG. 5 depicts graphically the results of an experiment in whichdiffering volumes (μL) of a suspension of fungal cells were added tourine to achieve cell concentrations in the range of about 5,000-35,000cells/ml urine; treated with alkaline and protease treatments as in FIG.3A, above; and then fungi in the sample were detected by staining withan MBP-conjugate as disclosed in EXAMPLE 8, below.

FIG. 6 depicts graphically the results of an experiment in whichdiffering volumes of a fungal cell suspension were added to 10 ml ofnormal urine to achieve cell concentrations in the range of about50,000-200,000 cells/ml urine; treated with alkaline and proteasetreatments as in FIG. 3A, above; and then fungi in the sample weredetected by staining with an MBP-conjugate as disclosed in EXAMPLE 8,below.

FIG. 7 depicts graphically the results of an experiment in whichdiffering volumes of a fungal cell suspension were added to a normalurine sediment to achieve cell concentrations in the range of about2,500-20,000 cells/ml of urine; treated with alkaline and proteasetreatments as in FIG. 3A, above; fungi were stained with anMBP-conjugate, and then stored at 4° C. for 3 days prior to analysis.The number of killed and denatured fungal particles was quantified asdisclosed in EXAMPLE 8, below.

FIG. 8 depicts graphically the results recorded in chemically treating,MBP-staining and cytofluorimetrically assaying aliquots of a urinesample from a patient with a urinary tract infection. The data areplotted as the number of killed and denatured fluorescent fungalparticles ("YG") counted per milliliter (mL) of urine per second againstthe different volumes of urine (mL) that were sedimented to obtain eachof the different respective samples, as disclosed further in EXAMPLE 8,below.

FIGS. 9A and 9B depicts graphically the results recorded in chemicallytreating, MBP-staining and cytofluorimetrically assaying a 1 mL aliquotof a urine sample from a patient with a bacterial urinary tractinfection. The data are plotted as the number of killed and denaturedfluorescent fungal particles against the fluorescence intensity(FSC-H/FSC-Height) of each particle. FIG. 9A shows all of the datapoints while FIG. 9B shows only those data points corresponding with aparticle size and particle fluorescence intensity that was appropriatefor yeast, i.e., `yeast gate` data, as disclosed further below inEXAMPLE 8.

FIGS. 10A and 10B depicts graphically the results recorded in chemicallytreating, MBP-staining and cytofluorimetrically assaying a 1 mL aliquotof a urine sample from a patient with a yeast urinary tract infection.The data are plotted as the number of killed and denatured fluorescentfungal particles against the fluorescence intensity (FSC-H/FSC-Height)of each particle. FIG. 10A shows all of the data points while FIG. 10Bshows only those data points corresponding with a particle size andparticle fluorescence intensity that was appropriate for yeast, i.e.,`yeast gate` data, as disclosed further below in EXAMPLE 8.

FIGS. 11A-D depicts graphically the results recorded in chemicallytreating, MBP-staining and cytofluorimetrically assaying a 1 mL aliquotof a urine sample from a patient with an unknown urinary tractinfection. The samples assayed in FIGS. 11A and 11B were collected fromthe patient one day prior to those assayed in FIGS. 11C and 11D (i.e.,day 1 and day 2). The data are plotted as the number of killed anddenatured fluorescent fungal particles against the fluorescenceintensity (FSC-H/FSC-Height) of each particle. FIGS. 11A and 11C showall of the data points collected on day 1 or day 2, respectively, whileFIGS. 11B and 11D shows only those data points corresponding with aparticle size and particle fluorescence intensity that was appropriatefor yeast, i.e., `yeast gate` data, as disclosed further below inEXAMPLE 8.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Documents cited herein by identification number, e.g. (1), are listed inthe Citations section which follows the Examples section, below.

In retrospect, few aspects of the present invention could have beenexpected based on the experience in the art at the time of theinvention. For instance, K_(a) 's for binding of native lysozyme tochitotriose are in the range of 1.1×10⁵ L/mol (3), 0.9-1.7×10⁵ L/mol(4); and for binding of catalytically inactivated lysozymes tochitotriose are in the range of 4.4×10⁵ L/mol (Gln³⁵), 4.0×10⁵ L/mol(Asp⁵²) (4). While chemically inactivated and mutant lysozymes, theirmethods of preparation, and their binding affinities had been disclosedpreviously, the compositions in the art were generally not suitable forpreparation of a diagnostic reagents for the following reasons: namely,

1. Catalytically active lysozymes degrade murein compounds;

2. Chemically inactivated lysozyme enzyme preparations were, in manycases, not of sufficient purity for use, and if pure, often containedactive enzyme as a contaminant and/or were not stable because theyunderwent regeneration of enzyme activity (5);

3. Catalytically inactive lysozyme reportedly binds non-specifically tohuman rbc, mouse rbc, chicken rbc, and Hep G2 cells as strongly as thelectin wheat germ agglutinin (6) creating an unacceptably highbackground;

4. Normal human serum, lymphocytes, monocytes and PMNs reportedlycontain endogenous chitinases (7) that could constitute confounding orinterfering substances in a diagnostic assay;

5. Lysozyme known to be present in biological samples, e.g., secretions,at high levels and is also a major cationic protein in the azurophil andspecific granules of PMN (7,11) which are released into inflammatorysecretions;

6. Bacterial and fungal cell wall components such as (GlcNAc-MurNAc)₂are known inhibitors of lysozymes (5; Ka 4.2×10³) and capable ofinhibiting these enzymes in a diagnostic assay format; and,

7. Bacterial LPS is a reported non-competitive inhibitor of lysozymewhich has a Ka for binding in the range of 10⁸ L/mol (11).

In consideration of these factors, mammalian cell debris in biologicalsamples would be expected to constitute an unacceptable high backgroundpreventing use of lysozymes and chitinases in diagnostic assays. Inaddition, bodily fluids would be expected to contain endogenouslysozymes and chitinases capable of acting as both potential interferingsubstances in diagnostic assay; and as hydrolases cleaving the analytesin a murein-binding diagnostic assay. Bacterial and/or fungal cell wallmaterials and biosynthetic products would also be expected to inhibitthe binding of a murein binding polypeptide to bacterial and/or fungalcell wall analyte. Thus, in retrospect, the disclosure in PCT/US92/02593(Tuse et al.; filed first in April of 1991 and without benefit ofpresent day understanding) was not justified in its propheticassumptions that chitinase (or lysozyme) could be used in diagnosticassays without significant and undue experimentation. Similarly, whileBenjaminson (9) and Chamberlain (8), which disclosed use ofF-NHS-chitinase for detecting chitin in histologic sections of planttissues, is not helpful in determining how one might proceed from aplant histochemical assay to a non-histologic diagnostic assay usefulwith biological samples of air, water, food, or bodily fluids. The Tuseinternational patent application (supra) did not apparently disclosecatalytically inactive murein binding proteins.

Binding of lysozyme and other murein binding polypeptides to denatured,fixed and killed bacterial and fungal particles is also believed to havebeen unknown at the time of the invention, including binding tobacterial murein compounds, or fungal murein-like compounds in deadbacterial and fungal particles after alcohol fixation (e.g., 80%ethanol), alkaline denaturation (e.g., 2M NaOH at 60° C. for 30minutes), and/or protease treatment (e.g., 0.25% trypsin at 37° C. for30 minutes), and/or treatment with periodate, N- or O-glycosidasesand/or treatment with acetic anhydride (as disclosed further below); or,that the subject binding with these denatured particles would be ofsufficient affinity (e.g., greater than nanomolar binding) and withlittle or no detectable cross-reactivity (e.g., with mammalian and plantfood substances), so as to enable use of the reagents prepared fromthese murein binding polypeptides in diagnostic assay formats.

The following properties of the instant in-situ binding enzyme-substrate(ISBES) methods were also believed to be unknown: namely, that treatingcertain types of biological samples could simultaneously effect thefollowing: namely, (i) killing infectious bacterial, fungal or viralagents in the sample to produce dead bacterial and fungal particles;(ii) lysing mammalian cells such as erythrocytes and leukocytes; (iii)disaggregating bacterial and fungal rafts into uniform singlecell/particle suspensions; (iv) dissociating bacterial and fungalparticles from other possible particulate materials in the samples suchas, plant cellulosic and lignin materials; (v) removing exterior fungalcell wall surface layers to expose murein-like compounds in the deadparticles; (vi) removing/denaturing mammalian cellular materials thatmight cross-react in the assay and contribute to nonspecific background;(vii) denaturing bacterial and fungal cell surface polypeptides toincrease susceptibility to protease treatments and/or to increaseaccessibility of murein-like ligands for binding to murein bindingpolypeptides; and, while (viii) retaining the ability of the deaddenatured bacterial and fungal particles to react with natural andmodified murein binding polypeptides.

Embodiments of the invention provide murein-binding polypeptides andpeptides (inclusively abbreviated MBP) that are capable of binding to awide variety of different bacterial mureins and fungal murein-likecompounds in a highly specific and saturable manner. While mureins arepresent in higher concentrations in gram-positive bacterial cell walls,they are also present (at lower concentrations) in gram-negative, othereubacteria and murein-like compounds are present in certain fungi. Thehighly sensitive and specific reagents and ISBES assay methods disclosedherein allow detection of bacterial murein compounds and murein-likefungal compounds even when present at relatively low concentrations.Preferably, the subject MBP have a binding affinity for diose, trioseand tetraose bacterial and fungal cell wall compounds that is in therange of about 10⁻³ L/mol to about 10⁻⁵ L/mol; and, the subjectcompounds are effective to bind the subject murein or murein-likecompound with a association constant (K^(a)) in the range of about5×10⁻⁵ L/mol to about 10⁻⁹ L/mol, preferably 5×10-7 L/mol to 10⁻⁹ L/mol,and most preferably greater than 10⁻⁹ L/mol. The subject pan-reactivemurein-binding proteins are used in a substantially purified form toformulate reagent preparations for use in diagnostic assays. Substantialpurity may be achieved e.g. by biochemical purification from naturalsources, or from selected bacterial, fungal, mammalian, avian, or insectsources; or, from the product of a genetically engineered expressionsystem. Preferably, the instant murein-binding polypeptides (orpeptides) are synthetic, modified, mutated, chemically inactivatedand/or genetically re-engineered non-antibody polypeptides (or peptides)that take their origin from a variety of different wild-typepolypeptides exhibiting an intrinsic binding affinities andspecificities for bacterial mureins. The subject wild typemurein-binding polypeptides suffer from certain disadvantages that maydiscourage their use in diagnostic assay formats, i.e. they may have abinding affinity for murein that are too low to encourage their use as apan-bacterial reagent; or, they may be cross-reactivity with otherbacterial carbohydrates and lack specificity; or, they may have a largeand cumbersome molecular size that discourages their use because of thecost of materials required to run an assay; or, they may becatalytically active and degrade mureins into soluble peptidoglycanproducts that are not longer detected in a diagnostic assay format.Representative examples of wild type catalytically active polypeptidesthat bind and cleave mureins include, but are not limited to, bacterial,fungal, mammalian, insect and avian bacterial cell wall degradingenzymes: e.g., muramidases, lysozymes, chitinases, β-glucosidases aswell as murein biosynthetic and degradative enzymes, (e.g. catalyzingthe reverse reaction.) Other representative examples of wild typeprecursors for the subject murein-binding proteins include lectins, andhybrid molecules, e.g., derived by recombinant or protein cross-linkingmethods. In general, wild-type (i.e., natural) murein-bindingpolypeptides are tested to determine whether their binding affinityfalls within the range requisite for use in an assay according to theinstant invention; in particular the subject wild-type murein bindingpolypeptides are tested to determine whether they will bind to killedand denatured bacteria and fungal particles according to the steps ofthe instant ISBES method (supra); and, whether the subject wild-typepolypeptides contain enzymatic activity and whether that activity can betolerated in an assay according to the instant ISBES methods (supra.)Preferably, the subject murein-binding polypeptides have less than 10%of the catalytic activity of a wild-type murein parental polypeptide.Most preferably, the subject murein-binding polypeptides have less than5% of the catalytic activity of a wild-type polypeptide. However, asillustrated in the Examples section below, the chemical treatment steps(supra) of the instant ISBES assay methods allowed the detection ofkilled and denatured bacterial and fungal particles in urine sedimentsusing a fluorescent conjugate of wild-type catalytically active hen eggwhite lysozyme.

Binding affinity (supra), choice of MBP (supra) and the steps of theinstant ISBES assay method (supra) are considered key factors inachieving success in the commercial diagnostic assays provided by theembodiments of the invention. Most preferably, embodiments of theinvention provide MBP compositions having binding affinities (K_(a)) inexcess than 10⁻⁹ L/mol, and most preferably MBP compositions that aresubstantially pure preparations consisting of MBP that have a bindingsite capable of binding several sugar residues constituting thepolysaccharide backbone (i.e., a spatial motif) of a murein ligand. Thesubject spatial motif preferably has about 4 to about 8 sugar residues,most preferably about 4 to about 6, with at least 2 of the residuesselected from among N-acetylglucosamine and N-acetylmuramic acid. Mostpreferably, the subject spatial motif has about 6 sugar residues, withat least 2 of the residues selected from among N-acetylglucosamine andN-acetylmuramic acid. Natural or catalytically inactivated hen egg whitelysozyme is a representative example of a murein binding compositionhaving a binding site that recognizes a 6 sugar spatial binding motif inmurein, and related compounds, and recognizes murein and murein-likecompounds in killed and denatured bacterial and fungal particlesprepared according to the instant ISBES assay methods. Catalyticallyinactive chitinase is one representative example of a murein bindingcomposition having a binding site that recognizes a 4 sugar spatialbinding motif in chitin and related compounds.

Embodiments of the invention provide, uniformity of compositions capableof conferring specificity and sensitivity on diagnostic assay. Thesubject compositions enable preparation of diagnostics reagents withinthe precise bounds necessary for regulatory compliance according toquality control and quality assurance protocols. The subjectcompositions have high binding affinity and advantageous stability atdifferent temperatures and on a storage shelf-life (i.e., shelf-lifestability.) In one presently preferred embodiment, catalyticallyinactive lysozyme is stable at acid pH (i.e., pH 4) and resists heatingat 100° C. at pH 4.5 for about 3 minutes. The subject high bindingaffinity and physical stability enables rapid commercially viable assayformats, i.e., requiring less than about 15 minutes to accomplishbinding between an MBP and an analyte in a biological sample, preferablyless than about 2 minutes to about 40 seconds, and most preferably about6 seconds to about 1 second. The subject high binding affinity MBP alsoprovides a favorable cost of goods, and lower cost-per-assay since onlysmall amounts of reagents and time are necessary to obtain an assayresult. Shelf-life stability of the subject liquid and powderedcompositions of the invention, often problematic in the diagnosticsindustry, was found to be excellent.

Embodiments of the invention provide diagnostic reagent solutions usefulfor detecting a murein compound in a biological sample according to oneor more of the instant assays. Reagent solutions are optimized tofacilitate specific binding interactions between the subject MBP and amurein ligand by choice of additives, buffers, stabilizers and the like.In one presently preferred embodiment, the subject reagent solutionswere optimized for use in the instant assays by adding (i) non-polarsolvents; and/or (ii) agents displacing water (e.g., polyethyleneglycol, sucrose, and the like); and/or (iii) detergents (e.g., NP 40) toa pH balanced buffer salt solution (e.g., Na⁺, K⁺, Li⁺ and the like) ofa MBP or MBP-SGC compound. MBP compounds having hydrophobic residuesinserted into the MBP protein, or having ionically, hydrophobically, andcovalently linked polyethylene (PEGylated) may exhibit increased mureinbinding activity.

Embodiments of the invention provide highly sensitive and specificnon-antibody diagnostic MBP reagents reactive with a wide range ofdifferent bacteria and fungi. The subject reagents consist of one ormore MBP, each capable of binding a murein compound present in aeubacteria, or a murein-like compound present in fungi and as thesubject compounds are present in a killed, fixed and treated biologicalsamples.

Other embodiments provide pan-reactive non-antibody MBP diagnosticassays useful for detecting bacteria and fungi in a biological samplethat has been fixed (e.g., in an organic solvent such as alcohol) orchemically treated (e.g., with an acid or base) to kill infectiousmaterials and thereby to reduce the risk to laboratory personnel.

Other embodiments provide non-antibody diagnostic reagents and assaymethods for: (i) distinguishing between bacteria and fungi in abiological sample that may contain plant and/or mammalian cellularmaterials; (ii) discriminating between fragments of bacteria or fungiand whole bacterial or fungal cells in a biological sample (i.e., basedon size); and, (iii) identifying and/or quantifying fungal cells in abiological sample containing bacterial cells, bacterial spores, fungalspores, or plant or mammalian cellular materials or fragments of anythereof. Representative assay methods for distinguishing, discriminatingand quantifying according to the instant assays methods are illustratedin the Examples section, below, and include use of low cytometry.

In other embodiments, the invention provides reagents and methods usefulfor (i) treating a fluid biological sample to increase the single cellcontent of a suspension of fungal cells; and, (ii) rendering the cellsuspension suitable for detecting and/or quantifying the extent ofcontamination of the sample, or the presence or severity of an infectionin a patient through the process of identifying and/or quantifying anumber of fungal cells in a defined volume of the subject biologicalsample. The instant reagents and methods are useful for treating fluidbiological samples that contain one or more of the following: namely,(i) fungal hyphae, (ii) rafts, aggregates or clumps of yeast cells,(iii) fungal cells in combination with bacterial cells or theirproducts, (iv) fungal cells in combination with plant cells or theirproducts, including plant cell wall materials, (v) fungal cells incombination with mammalian cells or their products, including mucins,lipids, lipopolysaccharides, glycolipids, proteoglycans and the like.Representative diagnostic reagents and assay methods are illustrated inthe Examples section, below.

In yet other embodiments, the invention provides a relativelyinexpensive source of MBP diagnostic reagents that are constitutivelyexpressed at high levels in relatively inexpensive bacterial, fungal,insect, mammalian and/or plant tissues, cells or cell cultures.

In other aspects, the invention provides simple diagnostic tests that donot require specialized personnel training or facilities, and useequipment that is commonly available in a clinical test laboratory,e.g., a clinical centrifuge, components supplied in the instant kit(below) with accompanying instruction.

Embodiments of the invention also provide pan-bacterial and pan-fungaldiagnostic reagents referred to herein as MBP-SGC conjugates, consistingof an MBP compound, preferably having a molecular size of about 55kilodaltons (KD) to about 12 KD, most preferably about 35 KD to about 12KD, that is linked through ionic, hydrophobic and covalent bonds to asignal generating compound (SGC.) The subject MBP-SGC conjugates have ahigh binding affinity for bacteria and fungi; a low catalytic activityfor murein substrates as they appear in killed and denatured bacterialand fungal particles according to the instant ISBES methods; and, have alow binding affinity for degraded soluble bacterial and fungal cell wallproducts. The subject MBP-SGC conjugates, contain a non-antibody MBPthat, unlike antibody reagents, does not contain an Fc portion capableof binding complement Clq, nor an antigen binding site capable ofcross-reacting with related antigens, nor a molecular size of 135-150KD, nor an antibody structure or charge distribution that can contributeto nonspecific electrostatic or hydrophobic binding interactions.

Embodiments of the invention provide in-situ binding enzyme-substrate(ISBES) assay methods for detecting and/or quantifying bacteria andfungi in biological samples. The instant methods include a step ofkilling bacteria and/or fungi in a biological sample by treatment with afixative, or alternatively, a chemical. The instant chemical treatmentpreferably is an alkaline treatment conducted at a temperature of about20° C. to about 100° C., preferably about 37° C. to about 100° C. andmost preferably at about 60° C. to about 70° C. The instant alkalinetreatment is conducted using a solution of a base at a concentration andfor a time effective to denature a bacterial or a fungal polypeptide.Representative examples of the instant fixatives include alcoholsolutions such as ethanol, methanol and isopropanol, as well as aldehydesolutions such as formaldehyde, paraformaldehyde and glutaraldehyde.Representative examples of the instant chemical base solutions so usefulinclude strong inorganic and organic bases such as about 0.5 M to about5 M sodium hydroxide; about 0.5 M to about 3 M potassium hydroxide; 1 Mto about 2.5 M ammonium hydroxide and the like. Preferably the chemicalbase solution is about 1 M to about 5 M sodium hydroxide, and mostpreferably about 2 M sodium hydroxide for use at the preferred and mosttreatment temperature and time (supra.) Illustrative concentrations andtreatment times effective to denature a fungal or bacterial polypeptideare disclosed in the Examples section below. "Denature" is intended tomean disrupting secondary and tertiary polypeptide structure involvede.g. in protein folding. Methods for determining protein denaturationare known in the art and include at least comparative scanningspectrophotometry e.g., in the UV and near UV, molecular sievechromatography, sucrose density gradient ultracentifugation, SDS-PAGEunder non-denaturing conditions (e.g., Osborne gels) and the like. Timeseffective to denature a fungal or bacterial polypeptide are dependentupon the temperature, determinable by one or more of the subjectmethods, and will generally be in the range of about 2 minutes to about30 minutes. The instant ISBES assay methods include assay formats inwhich detection or quantification of bacteria or fungi is conductedin-situ in fixed tissue samples by histochemistry; in samples of bodilyfluids such as urine and blood by flow cytometry or by enzyme-linkedsolid phase methods, machine assay methods or latex dipstick methods;and, in food, air, water samples by dot-blot methods. It has been foundthat the instant step of using a fixative or a chemical base to effectdenaturation of a bacterial or a fungal polypeptide, enables theoptional use of either a natural murein-binding polypeptide (i.e.,catalytically active and not genetically or chemically inactivated), ora catalytically disabled murein-binding polypeptide in the subject ISBESassay. Apparently, murein ligands in killed and denatured bacterial andfungal particles function as ligands for enzymatically active naturalpolypeptides but turnover of the subject substrates in the denaturedparticles is sufficiently slow to allow their use in certain ISBES assayformats. Those skilled in the art will recognize that comparativedeterminations can be made of natural and catalytically inactivemurein-binding polypeptides in a test ISBES assay format, and that adetermination can be made of whether the subject natural polypeptideshas performance characteristics allowing its use in an assay, e.g.performance including specificity, sensitivity, precision, background,cross-reactivity with non-murein compounds and reproducibility.Catalytically disabled murein binding polypeptides are preferred for usein ISBES assay formats, as disclosed further below.

In optional embodiments, the instant ISBES assay methods include a step,i.e., conducted after the fixative or alkaline treatment, of washing thedenatured biological sample to remove the fixative or chemical base, orneutralizing the base e.g. with HCl, and then treating with a solutionof a protease, a glycosidase, a sialidase or a periodate. The subjectsecond treatment step is effective to further reduce backgroundnon-specific signal generated in an ISBES assay and to increaseaccessibility of murein and murein-like compounds in bacterial andfungal cell wall for binding to the instant murein binding polypeptidereagents. One or more protease treatments hydrolyze bacterial and fungalcell surface proteins exposing murein and murein-like compounds, and atthe same time degrade mammalian and plant compounds which mightconstitute background reactivity in an assay. Representative proteasessolutions include trypsin at about 0.1% w/v to about 1% w/v, pronase atabout 0.25% to about 2% w/v, subtilisin at about 0.05% to about 1% andthe like. Preferably the protease solution is about 0.25% to about 1%trypsin. Glycosidases remove cell surface carbohydrates from bacterialand fungal cells, exposing mureins and murein-like compounds anddegrading potential mammalian and plant crossreacting materials whichmight contribute to background in an assay. Representative glycosidasesolutions include both N- and O-glycosidases at concentrations of about50 μg/ml to about 10 mg/ml. Sialidases remove charged sialic acid fromcell surface carbohydrates in mammalian and eubacterial and fungalcells. The resultant reduction in cell-surface charge may decreasenon-specific background in an assay. Representative sialidases includecommercially available N-acetyl neuraminidases from several sources(Sigma Chemical Co., St. Louis, Mo.) Periodate removes sugar residues atvicinal hydroxyl-groups, increases availability of murein-like compoundsin fungi for binding to MBP, and decreases background reactivity withcomplex carbohydrates synthesized by mammalian cells, e.g.,erythrocytes, leukocytes, tissue cells, tumor cells and the like.Representative periodate solutions include sodium periodate at aconcentration of about 0.1% to about 1%. In yet other optionalembodiments, the instant second treatment step may include sequentialtreatments with one or more chemical base, one or more protease, one ormore glycosidase and/or the periodate, e.g., 1M KOH treatment followedby an optional protease treatment to expose murein and murein-likecompounds in bacteria and fungi in histopathologic tissue sectionscollected from infected animals (i.e., man and domestic animals), aswell as, for solubilizing potentially cross-reactive mammalian tissuematerials.

In yet other optional embodiments, the instant ISBES assay methodsinclude a step, i.e., conducted after the fixative or alkaline treatmentor after the instant second step, of washing the denatured biologicalsample to remove any fixative, chemical base or second treatment stepcompound, and then treating with a solution of an anhydride effective toaccomplish N-acetylation of carbohydrate compounds in the denaturedbacterial or fungal particles. The subject N-acetylation step iseffective to increase the sensitivity, e.g., number of bacteria or fungirequired for identification or quantification, of an instant ISBES assayprobably by increasing the binding of a murein-binding polypeptide withthe a murein or murein-like compound in the subject killed and denaturedfungal or bacterial particle. Most preferably, the means forN-acetylation consists of conditions suitable for N-acetylation ofpolysaccharides, e.g., in a suitable buffer pH 8. Representativeexamples of anhydride solutions so useful include about 2% (v/v) toabout 5% (v/v) acetic anhydride in sodium bicarbonate buffer at pH 8,and about 0.2 M to about 0.5 M acetyl chloride in sodium bicarbonatebuffer at pH 8.

In other embodiments the invention provides reagents and methods forconducting rapid high through-put continuous-flow cytometric assays. Inone presently preferred embodiment, the subject cytometry assay is acytofluorimetric assay, and most preferably the subject cytofluorimetricassay is capable of quantifying a number of bacterial or fungal cells ina biological sample collected from a subject in need thereof, thereby todetermine the presence and/or severity of an infection in the subject.In another presently preferred embodiment, the subject cytometry assayis a cytofluorimetric assay, and most preferably the subjectcytofluorimetric assay is capable of quantifying a number of bacterialor fungal cells in a biological sample collected from food, water orair, thereby to determine the presence and/or amount of contamination inthe sample. In an alternative preferred embodiment, the subjectcytofluorimetric assay consists of a simultaneous assay format havingthe steps of (i) binding a non-antibody MBP-SGC conjugate to a bacterialor a fungal cell; (ii) discriminating between the bacterial and thefungal cell, or degradative products of these cells, (e.g. by size orfluorescence intensity), thereby to identify the microbial source(bacterial or fungal) of an infection in a subject in need thereof. Inyet another alternative preferred embodiment, the subjectcytofluorimetric assay provides a rapid method for determining theantibiotic sensitivity of a bacteria or a fungi in a biological sample.The subject assay involves the steps of (i) quantifying the number ofbacterial or fungal cells in a biological sample; (ii) culturing analiquot of the biological sample for a period of time and underconditions suitable for growth of the bacteria or the fungus in thepresence or absence of an antibiotic; and, (iii) determining thebacteria or fungi to be antibiotic sensitive if growth occurs in theabsence of antibiotic but not in the presence of antibiotic.Representative times for culture of biological samples containingbacteria are about 2 to about 6 hours, preferably about 2 to about 4hours, and most preferably about 2 hours. Representative times forculture of biological samples containing fungi are about 2 hours toabout 20 hours, preferably about 2 hours to about 8 hours and mostpreferably about 3 hours to about 6 hours.

Yet other embodiments of the invention provide murein bindingpolypeptide reagents and ISBES assay methods that offer cost-savings andother commercial advantages (e.g., shelf life stability) formanufacturers and users of diagnostic test reagents, kits and reagentpackages marketed for detection of bacterial and fungal infections.Representative examples of the subject assay methods are provided belowin the Examples section below.

Embodiments of the invention provide murein-binding polypeptidesprepared for use as pan-bacterial and pan-fungal diagnostic reagents bychemically conjugating the subject MB-polypeptide to a signal generatingcompound. Representative signal generating compounds are disclosedbelow, and illustrated in the Examples section.

The subject murein binding polypeptide as it is conjugated with aparticular signal generating compound may optionally be linked throughan additional chemical linking group to a magnetic particle, or tocapture compounds such as biotin and avidin.

Embodiments of the invention provide in-situ binding, enzyme-substrate(abbreviated herein ISBES) assay methods, disclosed herein, employmurein-binding polypeptide reagents to identify bacteria and fungiin-situ, i.e. without isolation and culture. For example, the reagentsmay be used to detect bacteria or fungi in-situ in fixed tissue samplesby histochemistry; in samples of bodily fluids such as urine and bloodby flow cytometry or by enzyme-linked solid phase methods, machine assaymethods or latex dipstick methods; and, in food, air, water samples bydot-blot methods. ISBES sample preparation methods include chemicallytreating certain types of biological samples to simultaneously effectthe following: namely, (i) killing infectious bacterial, fungal or viralagents in the sample to produce dead bacterial and fungal particles;(ii) lysing mammalian cells such as erythrocytes and leukocytes; (iii)disaggregating bacterial and fungal rafts into uniform singlecell/particle suspensions; (iv) dissociating bacterial and fungalparticles from other possible particulate materials in the samples suchas, plant cellulosic and lignin materials; (v) removing exterior fungalcell wall surface layers to expose murein-like compounds in the deadparticles; (vi) removing/denaturing mammalian cellular materials thatmight cross-react in the assay and contribute to nonspecific background;(vii) denaturing bacterial and fungal cell surface polypeptides toincrease susceptibility to protease treatments and/or to increaseaccessibility of murein-like ligands for binding to murein bindingpolypeptides; and, while (viii) retaining the ability of the deaddenatured bacterial and fungal particles to react with natural andmodified murein binding polypeptides.

Embodiments of the invention also provide quantitative and qualitativeflow cytometric and cytofluorimetric ISBES methods for determining thepresence, severity and type of microbial infection in a patient bydetecting and quantifying the number of bacterial and/or fungi in abiological sample which are reactive with a murein binding polypeptidereagent. The latter particle size and light scattering ISBES methodsallow rapid quantification and/or discrimination between bacteria andfungi in patient samples, and are not effected by the presence of cellwall fragments.

Embodiments of the invention provide kits for identifying eubacteria andfungi in samples of air, water and biological samples. Representativeexamples of the subject kits are contained within a box that has a setof instructions and an opening for one or more containers (e.g. bottles,reagent packages and the like) for holding the following solutions orpowders: namely, (i) a solution or powder comprising a murein bindingpolypeptide conjugate having a signal generating compound and a buffer;(ii) an optional solution for resuspending the subject conjugate; (iii)a solution of a chemical base compound, for killing bacteria and fungiand denaturing a bacterial or a fungal polypeptide; (iv) an optionalsolution for neutralizing the base; (v) an N-acetylation buffer andreagent; (vi) an optional solid phase MBP-capture (as disclosed furtherbelow and illustrated in the Examples section); wherein, all of thesubject reagents are present in the kit in a form suitable for use in anISBES assay method conducted according to the steps of the instructionsincluded within the kit.

As used herein the symbols for amino acids are according to theIUPAC-IUB recommendations published in Arch. Biochem. Biophys. 115:1-12,1966 with the following single letter symbols for the amino acids:namely,

    ______________________________________                                        L, Leu,  V, Val, Valine                                                                            Y, Tyr,   D, Asp, Aspartic Acid                          Leucine              Tyrosine                                                 I, Ileu, P, Pro, Proline                                                                           W, Trp,   E, Glu, Glutamic Acid                          Isoleucine           Tryptophan                                               M, Met,  G, Gly, Glycine                                                                           N, Asn,   K, Lys, Lysine                                 Methionine           Asparagine                                               T, Thr,  A, Ala, Alanine                                                                           Q, Gln,   R, Arg, Arginine                               Threonine            Glutamine                                                F, Phe,  S, Ser, Serine                                                                            C, Cys,   H, His, Histidine                              Phenylalanine        Cysteine                                                 ______________________________________                                    

The symbols for protective groups used in the synthetic process aredescribed in Schrodex and Lubke, "The Peptides", Academic Press, N.Y.1965, e.g., Boc, t-butyloxycarbonyl and Bzl, benzyl. Other abbreviationsinclude: e.g., HPLC, high pressure liquid chromatography; TFA,trifluoroacetic acid; K_(D), dissociation constant, Ka, associationconstant; Keq, equilibrium constant; kcat, enzyme catalytic constant;Km, Michaelis Menten enzyme constant; V_(max) maximal theoretical enzymevelocity; FITC, fluorescein isothiocyanate; RITC, rhodamineisothiocyanate; F-NHS, N-hydroxy-succinimidyl-fluorescein; HRP, horseradish peroxidase; AP, alkaline phosphatase; Ac, for an acetyl group;Gal, for a galactosyl group; Glu, for a glucosyl group; GlcNAc,N-acetylglucosamine; Glu, glucose; MB, murein binding; MBP, for mureinbinding polypeptide (or peptide); kD, kilodaltons molecular size;kcat/Km, a measure of enzyme catalytic efficiency expressed as thecatalytic constant divided by the Km; PBS, phosphate buffered saline;ELMBA, enzyme-linked murein binding assay; ELISA, enzyme-linkedimmunosorbent assay (not an object of the invention); and, FMBA,fluorescent murein binding assay; SGC, signal generating compound.

Terms used herein are intended to have meaning as follows: namely,

"Eubacteria" is intended to mean a bacteria having a murein compounddetectable using the reagents and/or methods of the invention asdisclosed further below, and as relying in particular upon a diagnosticreagent containing a murein binding polypeptide (or peptide)-conjugateaccording to the instant definitions and accompanying disclosure.Representative eubacteria include: gram negative bacteria, gram positivebacteria, mycobacteria, spores from bacteria, and the like.Representative eubacteria in biological fluids (as defined below)include: chlamydia, toxoplasma, staphylococci, streptococci, gonococci,pneumococci, and the like. Representative eubacteria in biologicalsamples of air (as defined below) include Legionella, spores of anthrax,and the like. Representative eubacteria in food samples includestaphylococci, E. coli, Clostridium and the like.

"Fungi" is intended to mean a eukaryotic cell having a nuclear membraneand cell wall. The subject fungi may grow as single cells (e.g.,yeasts), chains (e.g., hyphae), aggregates, rafts and the like, and arenot plant or mammalian cells. The subject fungi contain a murein-likecompound capable of binding an MB polypeptide or peptide as e.g.detectable using the reagents and/or methods of the invention (disclosedfurther below.) Representative examples of murein-like compounds thatmay be detected by the subject diagnostic reagents include fungalcompounds containing N-acetyl-glucosaminoglucans. Representative fungiinclude: Aspergillus sp., Candida sp., Cryptococcus sp., Histoplasmasp., Coccidiomycosis sp., Sacchromyces sp., Blastomyces sp.,Actinomycetes sp. and the like.

"Murein" is intended to mean a macromolecular cross-linked cell wallassembled by eubacteria from several individual linear unbranchedmuropolysaccharide backbones each consisting of both of the amino sugarsN-acetylglucosamine and N-acetylmuramic acid. The subject severalmuropolysaccharide backbones are cross-linked together by amide bondsformed between carboxylic acid groups in muramic acids and shortoligopeptide side chains and peptide bonds formed between adjacentoligopeptide side chains. The subject cross-linked compounds form 2- or3-dimensional networks. The subject murein compounds are not smallpolysaccharide chains such as dioses, trioses or tetraoses.Representative mureins are commonly found in higher concentrations ingram-positive bacteria, but are also present (albeit in somewhat lesseramounts) in gram-negative bacteria, mycobateria, bacterial spores, fungiand the like. The subject murein compounds, when present in biologicalsamples under measurement in the instant assays are referred to hereinas "analytes".

"Murein-like" compounds is intended to mean a macromolecularcross-linked cell wall compound assembled by a eubacteria or a fungifrom several individual unbranched glycosyl backbones consisting ofeither N-acetylglucosamine or N-acetylmuramic acid. The subjectmurein-like compounds are not small polysaccharide chains such asdioses, trioses or tetraoses. Further, the subject compounds aredistinguished by their ability to bind a murein-binding polypeptide andto function as an analyte in an assay according to the instantinvention.

"Analyte" is used herein to refer to a compound present in a biologicalsample whose determination is of interest to a user of the instantmethods, wherein the subject analyte contains at least one mureincompound (supra) or one murein-like compound (supra.) Representativeexamples of analytes include intact fungal and eubacterial cells,secreted bacterial and fungal cell wall murein compounds (supra),degradative products thereof such as cell wall fragments, peptidoglycanscomplexes in solution, and the like.

"Chitin" is used herein to refer to cross-linked unbranched chainscomposed of only β-(1,4)-2-acetamido-2-deoxyglucose, also known asN-acetyl-D-glucosamine.

"Murein binding polypeptide" is used interchangeably with theabbreviation "MB-polypeptide" to refer to a polypeptide that is composedof about greater than about 5000 molecular weight and consisting ofamino acids arranged in a serial array with each amino acid peptidebonded to its neighboring amino acid in a secondary structure.Preferably, the subject MBP have a binding affinity for diose, trioseand tetraose bacterial and fungal cell wall compounds that is in therange of about 10-³ L/mol to about 10-⁵ L/mol and are effective to bindthe subject murein or murein-like compound with a association constant(K_(a)) in the range of about 5×10-⁵ L/mol to about 10-⁹ L/mol,preferably 5×10-⁹ L/mol to 10-⁹ L/mol, and most preferably greater than10-⁹ L/mol. Representative examples of murein-binding wild-typepolypeptides that may prove suitable for preparing murein-bindingpolypeptide non-antibody diagnostic reagents include, e.g.: (i) enzymescapable of hydrolyzing a murein substrate, e.g. selected from amonglysozymes (as disclosed in EXAMPLE 1, below); (ii) lectins binding to amurein; (iii) bacterial enzymes involved in bacterial cell wallsynthesis, turnover and/or degradation; (iv) fungal or viral enzymesinvolved in murein degradation (e.g., Pseudomonas autolysins,bacteriophage T4 lysozyme); and (v) peptidases having specificity formurein (e.g., an alanyl amidase). Preferably, the subject enzymes arecatalytically disabled to decrease hydrolysis of the instant mureincompound bound thereto. Most preferably, the subject enzymes are eitherchemically altered (e.g., EXAMPLE 3, below) or catalytically disabledmutant enzymes, (e.g., recombinantly derived enzymes such as in EXAMPLE1, below.) The subject most preferred chemically altered or mutantenzymes retain murein binding ability but lack more than about 70% toabout 95%, preferably more than about 85% to about 90%, and mostpreferably more than about 90%, of their catalytic activity.Representative examples of enzymes that may be so-modified according tothe instant invention include those disclosed in TABLE 1, below: e.g.,acetyl-muramoyl-D,L-Alanyl amidases, bacterial cell wall penicillinbinding proteins, Alanyl D,D- or D,L-endopeptidases, D,D- orD,L-carboxypeptidases, transglycosyl transferases, peptidyltransferases, muramoyl isomerases; muramoyl transglycosylases, mureinautolysins, murein hydrolases, β-glucosidases, lysozymes and the like.Preferably, the subject murein-binding polypeptides have amino acidsequences capable of ligand binding (as defined below), wherein thepresently most preferred ligand is a murein ligand as isolated from akilled and denatured bacterial or fungal particle following any of thefollowing treatments: namely, (i) fixation with 80% ethanol, (ii)alkaline treatment with 2M NaOH at 60° C. for about 15 minutes to about30 minutes, (iii) the subject alkaline treatment followed by an optionalprotease treatment with 0.25% trypsin at 37° C. for 30 minutes, and/or(iv) the subject alkaline treatment followed by an optionalperiodate-treatment, and/or (v) the subject alkaline treatment followedby a treatment with acetic anhydride (i.e., wherein the subjecttreatment conditions are in accord with the instant disclosure andillustrations.) Most preferably, the binding interaction between thepreferred MBP and a denatured ligand, i.e., according to any of thesubject treatments, occurs with high affinity and specificity, and witha binding affinity that is greater than nanomolar (as defined andillustrated further below.) Most preferably, the binding interactionbetween the most preferred murein binding polypeptides and one of thesubject denatured bacterial or fungal ligands, is not inhibited by100-fold molar excess of a mammalian or plant ligand such as might bepresent in a food substance.

Representative examples of non-antibody proteins having amino acidsequences and functional properties rendering them suitable candidatemurein binding polypeptides include murein biosynthetic and hydrolyticenzymes produced by mammals, insects, bacteria, bacteriophage and fungi,some representative examples of which are set forth in TABLE 1, below.

                                      TABLE 1                                     __________________________________________________________________________    Representative Candidate Murein Binding Polypeptides                          Murein Binding                     Enzyme    Doc. ID                          Polypeptide                                                                           Source   Illustrative Characteristics                                                                    Activity  No.*.sup./b                      __________________________________________________________________________    Autolysins                                                                             P. aeruginosa                                                                         26 kD membrane vesicle hydrolase                                                                N--Ac-muramoyl-L-                                                                       14                                                                  Ala amidase                                        P. aeruginosa                                                                          15-19 kD extracellular hydrolase                                                                amidase   32                               Cell wall                                                                             Bacterial cell                                                                         membrane proteins binding beta lactam                        metabolic                                                                             walls    antibiotics; enzymes involved in                             enzymes; e.g.,   synthesis of murein:                                         penicillin                                                                            Strep. pneumoniae                                                                      penicillin binding protein 2 (PBP2)                                                             transpeptidase                                                                          15                               binding proteins                                                                      E. coli  PBP2              transpeptidase                                                                          25                                       E. coli  penicillin binding protein 7/8 (PBP7/8)                                                         Ala DD-endopeptidase                                                                    21                                                PBP3              transferase                                                                             34                                       E. coli  PBP4: 477 amino acids                                                                           DD-carboxypeptidase                                                                     29                                       E. coli  Membrane lytic transglycosylase, 38 kD                                                          transglycosylase                                                                        19.sup.r,22                              several  muramidases                 19.sup.r                                 several  chitinases                  19.sup.r                                 several  glycosyltransferases                                                 Sal. typhi                                                                             lipoprot. glyceryl transferase 1gt, 291                                                         glyceryl transferase                                                                    23                                       E. coli  70.5 kD Slt transglycosylase (EC3.2.1-)                                                         transglycosylase                                                                        28                                       E. coli  mepA penicillin-insensitive                                                                     endopeptidase                                                                           30                                       E. coli  EC3.5.1.28 amidase                                                                              N--Ac-mur-Ala                                                                           35                                       E. coli  gene product mltB, 37 kD protein                                                                amidase   18                                       L. monocytogenes                                                                       p60 murein hydrolase                                                                            murein hydrolase                                                                        20                                                                  murein hydrolase                           Cell wall                                                                             Bacteria with cell                                                                     Enzymes required for induction of                            recycling                                                                             walls, esp. species                                                                    penicillin resistance                                                                           several   16.sup.r                         enzymes with penicillin                                                                        Amp C beta-lactamase                                                                            LD-endopeptidase                                                                        26                                       resistant serotypes                                                                    32 kD norcardicin A sensitive peptidase                                                         LD-carboxypeptidase                                                                     27                               bacteriophage                                                                         T4 in E. coli                                                                          protein T, 18 kD  lysozyme  17,33-40                                 HB-3 in Strep.                                                                         36 kD hbl gene product murein hydrolase                                                         amidase   24,36                                    Dp-1 in Strep.                                                                         endo-N--Ac-muramyl-L-Ala amidase                                                                endopeptidase                                                                           31                                                                            32                               Commercially                                                                          Sigma Chem. Co.,                                                                       EC3.2.1.17        hen lysozyme                                                                            #L6876                           available                                                                             St. Louis, MO                                                                          carboxy-methylated-maleylated reduced                                                           lysozyme-cat.inactive.sup.a                                                             #L1526                           enzymes                                      #C7809                                            EC3.2.1.21                  #C6137                                            EC3.5.1.11        β-glucosidase                                                                      #G4511                                                              penicillin amidase                                                                      #P3319                           __________________________________________________________________________     *.sup.r = review; kD, kilodalton molecular size, Doc. ID No., see citatio     list following the examples section;                                          .sup.a cat. inactive                                                          .sup.b catalycally inactive                                              

It is believe that prior to the instant invention the value of mureinbinding proteins as diagnostic reagents was not appreciated noradequately disclosed because many exhibit hydrolytic activity consideredincompatible with performance in a diagnostic assay, and/or low bindingaffinity, and/or apparent lack of specificity for murein compounds(supra). The subject murein binding polypeptides are distinguished fromchitinases in the manner set forth in TABLE 2, below.

                  TABLE 2                                                         ______________________________________                                                   Ligand                                                             Polypeptide  Murein Compound                                                                            Chitin Compound                                     ______________________________________                                        MB-Polypeptide                                                                             +            +                                                   Chitinase    -            +                                                   ______________________________________                                         +, represents a association constant for binding that is less than about      × 10-.sup.5 L/mol;                                                      -, represents a association constant for binding that is greater than         about 5 × 10-.sup.5 L/mol, e.g., 10-.sup.4 L/mol.                  

"Lysozyme" is intended to mean a muramidase capable of catalyzinghydrolysis of a bond in an N-acetyl-muramoyl compound. Representativelysozymes include those in IUB class E.C.3.2.1.17, e.g., hen egg whitelysozyme, human lysozyme, bacteriophage lysozymes, and other mammalian,animal, plant, fungal, bacterial, protist, viral or bacteriophagelysozymes.

"β-glucosidase" is intended to mean a β-D-glucosidase glucohydrolase.Representative examples of glucosidases may be found in IUB classEC3.2.1.21.

"Murein binding peptide", abbreviated "MB peptide" is used to refer to apeptide that is composed of about 50 to about 20 amino acids, preferablyless than about 20 amino acids, that are arranged in a serial array witheach amino acid peptide bonded to its neighboring amino acid in asecondary structure. The constituent amino acids are linked in serialarray to form a stable secondary structure that may be furtherstabilized by chemical modifications designed to create a stabletertiary structure. The subject modified stable MB peptide retains thethree dimensional array of amino acids contained in the active site ofan enzyme capable of hydrolyzing a murein, but the subject MB peptidewhile retaining the capacity for binding is catalytically inactive, i.e.according to the definition appearing below. In one representativeexample, the subject stable secondary structure of the MB peptideconsists of a stable alpha helical tertiary structure, and in this casethe amino acids utilized for the modification are preferably selectedfrom among the group of amino acids previously termed "helix-formers" byChou and Fasman (12; at Table 1, page 51), the list of amino acidsstanding as modified and subject to the limitations discussed in O'Neiland DeGrado (13; at Table 2, page 650), the disclosures of bothdocuments being incorporated herein by reference. As referred to herein,helix formers is intended to mean both weak ("h.sub.α ") and strong("H.sub.α ") helix formers as set forth in Chou and Fasman, supra, andas also conforming with amino acids having P α values >1.0 at set forthin O'Neil and DeGrado (13; at Table 2, page 650). Representativehelix-formers are Ala (A) and Leu (L) which are recognized in the art asstrong helix formers. A comparative ranking of helix forming amino acidsfrom Chou and Fasman, supra and O'Neil and DeGrado (13), supra isprovided in TABLE 3, below.

                  TABLE 3                                                         ______________________________________                                        Helix-Forming Amino Acids                                                     Rank order of Chou Rank order of                                              and Fasman         O'Neil and DeGrado                                         (H.sub.α  and h.sub.α)                                                               (P.sub.α > 1.0)                                      ______________________________________                                        E (H.sub.α)  A (1.6)                                                    A (H.sub.α)  L (1.5)                                                    L (H.sub.α)  F (1.45)                                                   H (h.sub.α)  M (1.44)                                                   M (h.sub.α)  W (1.34)                                                   Q (h.sub.α)  I (1.31)                                                   W (h.sub.α)  R (1.25)                                                   V (h.sub.α)  Q (1.22)                                                   F (h.sub.α)  E (1.18)                                                   --                 V (1.09)                                                   --                 K (1.05)                                                   --                 D (1.03)                                                   ______________________________________                                    

Representative examples of non-helix forming amino acids include Pro(P), Gly (G), Tyr (Y), referred to in the art as helix-breaking aminoacids, and these residues are commonly found in helix side-caps adjacentto, but not in, regions of alpha helical structure. Representativeregions of helical sequence motifs in murein binding polypeptidesinclude those the substrate binding sites and catalytic sites ofmuramidases, and transglucosylases including those disclosed previouslyin Dijkstra et al. (2). The subject MB peptides may be synthesized, e.g.by organic synthesis (below) of a murein binding site motif, oralternatively, molecular mimetics may constructed using branched andstraight hydrocarbon (olefin) chains to achieve spacing between organicresidues capable of mediating murein binding.

"MBP" is used herein as an interchangeable, and inclusive, as anabbreviation for a murein binding polypeptide or a murein bindingpeptide. "Endogenous MBP" is used herein to mean that the subject MBPcompound is bound to a murein compound (i.e., an analyte) in abiological sample (defined below) as the subject sample is collectedfrom a source, e.g., air, biological fluid and the like. As such, anendogenous MBP could conceivably constitute a confounding substance inan assay according to the invention.

"Murein binding diagnostic reagents" is intended to mean a reagentsuitable for use in a test assay for identifying a eubacteria or a fungi(e.g., a yeast) in a biological sample, e.g. a patient sample or asample of a sample of food, water or air. The subject murein-bindingreagent is provided in a reagent suitable for binding an analyte underconditions maximizing the subject binding interaction while minimizingcross-reactivity with any plant, mammalian, or avian tissue that may bepresent in the biological sample. The latter specific bindinginteraction is of course of considerably value when the subjectbiological sample is collected from a food substance, e.g., plant,animal or avian tissues. The subject murein binding diagnostic reagentcommonly contains: (i) an MBP linked to a "signal generating compound",i.e., a "conjugate" (as defined supra); (ii) one or more buffers,additives, excipients and the like for stabilizing and preserving thesubject MBP-conjugate during storage; and/or, one or more substances forpromoting the binding activity of the subject MBP-conjugate to a mureinin a test assay.

"Signal generating compound", abbreviated "SGC", is intended to mean amolecule that can be linked to a MBP (e.g. using a chemical linkingmethod as disclosed further below and is capable of reacting to form achemical or physical entity (i.e., a reaction product) detectable in anassay according to the instant disclosure. Representative examples ofreaction products include precipitates, fluorescent signals, compoundshaving a color, and the like. Representative SGC include e.g.,bioluminescent compounds (e.g., luciferase), fluorophores (e.g., below),bioluminescent and chemiluminescent compounds, radioisotopes (e.g., ¹²⁵I, ¹⁴ C, ³ H and the like), enzymes (e.g., below), binding proteins(e.g., biotin, avidin, streptavidin and the like), magnetic particles,chemically reactive compounds (e.g., colored stains),labeled-oligonucleotides; molecular probes (e.g., CY3, ResearchOrganics, Inc.), and the like. Representative fluorophores includefluorescein isothiocyanate, succinyl fluorescein, rhodamine B,lissamine, 9,10-diphenylanthracene, perylene, rubrene, pyrene andfluorescent derivatives thereof such as isocyanate, isothiocyanate, acidchloride or sulfonyl chloride, umbelliferone, rare earth chelates oflanthanides such as Europium (Eu) and the like. Representative SGCuseful in an MBP-conjugate include the enzymes in: IUB Class 1,especially 1.1.1 and 1.6 (e.g., alcohol dehydrogenase, glyceroldehydrogenase, lactate dehydrogenase, malate dehydrogenase,glucose-6-phosphate dehydrogenase, glyceraldehyde-3-phosphatedehydrogenase and the like); IUB Class 1.11.1 (e.g., catalase,peroxidase, amino acid oxidase, galactose oxidase, glucose oxidase,ascorbate oxidase, diaphorase, urease and the like); IUB Class 2,especially 2.7 and 2.7.1 (e.g., hexokinase and the like); IUB Class 3,especially 3.2.1 and 3.1.3 (e.g., alpha amylase, cellulase,β-galacturonidase, amyloglucosidase, β-glucuronidase, alkalinephosphatase, acid phosphatase and the like); IUB Class 4 (e.g., lyases);IUB Class 5 especially 5.3 and 5.4 (e.g., phosphoglucose isomerase,trios phosphatase isomerase, phosphoglucose mutase and the like.) Thesubject signal generating enzymes may be coupled to a non-antibody MBP,e.g., lysozyme, or to a second binding partner used in combination witha MBP, e.g., a different MBP (i.e., MBP #2; as disclosed further,below.) The subject SGC share the common property of allowing detectionand/or quantification of a murein analyte in a test sample. Preferably,the subject signal generating compounds are detectable using a visualmethod, a spectrophotometric method, an electrical method (e.g., achange in conductance, impedance, resistance and the like), or, afluorescent detection method.

"Solid phase", as used herein, is intended to mean a surface to whichone or more reactants may be attached electrostatically,hydrophobically, or covalently. Representative solid phases includee.g.: nylon 6; nylon 66; polystyrene; latex beads; magnetic beads; glassbeads; polyethylene; polypropylene; polybutylene; butadienestyrenecopolymers; silastic rubber; polyesters; polyamides; cellulose andderivatives; acrylates; methacrylates; polyvinyl; vinyl chloride;polyvinyl chloride; polyvinyl fluoride; copolymers of polystyrene;silica gel; silica wafers glass; agarose; dextrans; liposomes; insolubleprotein metals; and, nitrocellulose. Representative solid phases includethose formed as beads, tubes, strips, disks, filter papers, plates andthe like. Filters may serve to capture analyte e.g. as a filtrate, oract by entrapment, or act by covalently-binding MBP onto the filter(e.g., EXAMPLE 3, below). According to certain embodiments of theinvention, a solid phase capture reagent for distribution to a user mayconsist of a solid phase (supra) coated with a

"capture reagent" (below), and packaged (e.g., under a nitrogenatmosphere) to preserve and/or maximize binding of the capture reagentto a murein analyte in a biological sample.

"Capture reagent" is intended to mean an immobilized murein bindingpolypeptide (or peptide) capable of binding a murein compound ormurein-like compound. The subject capture reagent may consist of asolution or MBP modified so as to promote its binding to a solid phase,or as an MBP already immobilized on a solid phase, e.g., immobilized byattaching the MBP to a solid phase (supra) through electrostatic forces,van Der Waals forces, hydrophobic forces, covalent chemical bonds, andthe like (as disclosed further below.) Representative examples ofMBP-capture reagents are disclosed (EXAMPLE 1, below) and include mobilesolid phase MBP-capture reagents such as MBP immobilized on movablelatex beads e.g. in a latex bead dipstick assay.

"Detect reagent" is intended to mean a conjugate containing an SGClinked to a murein binding polypeptide (or MB peptide). Representativeexamples of the instant detect reagents include SGC-MBP; complexes ofantibiotic compounds with MBP, i.e., at a site distinct from the mureinbinding site, thereby forming a sandwich; antibodies (Ab) capable ofspecifically binding to an MBP and conjugated with a SGC, e.g., SGC-Ab,and the like. The subject detect reagents include mobile solid-phasedetect reagents such as movable latex beads in latex bead dipstickassays.

"Biological sample" is intended to mean a sample obtained from a living(or dead) organism, e.g., a mammal, fish, bird, reptile, marsupial andthe like. Biological samples include tissue fluids, tissue sections,biological materials carried in the air or in water (e.g., bacteria,fungi, spores and the like) and collectable therefrom e.g., byfiltration, centrifugation, and the like. Representative biologicalfluids include, e.g. urine, blood, plasma, serum, cerebrospinal fluid,semen, lung lavage fluid, feces, sputum, mucus, water carryingbiological materials and the like. Representative biological samplesalso include foodstuffs, e.g., samples of meats, processed foods,fishes, cereal grains and the like. Biological samples also includecontaminated solutions (e.g., contact lens solution, irrigationsolutions, intravenous solutions and the like), contaminated humanhealthcare products (e.g., shampoos, toiletries, contact lenses, and thelike), swab samples from food preparation facilities (e.g., restaurants,slaughter-houses, cold storage facilities, supermarket packaging and thelike). Biological samples may also include in-situ tissues and bodilyfluids (i.e., samples not collected for testing), for example, theinstant ISBES methods may be useful in detecting the presence orseverity or bacterial infection in eyes (e.g., usingMB-polypeptide-latex eye drops); or, the presence or extent ofcontamination of carcasses e.g. in a packing plant. Thus, embodiments ofthe invention provide ISBES methods useful in testing a variety ofdifferent types of biological samples for the presence or amount of abacterial or fungal contamination or infection.

"Ligand" as used herein refers to a murein compound capable of bindingto an MBP binding site. Representative examples of ligands includemurein-containing complex cell wall compounds (supra) as found in avariety of gram positive and gram negative bacteria and murein-likecompounds found in fungi (defined supra.) The subject ligand is capableof filling a three-dimensional space in binding site of a MBP so thatelectrostatic repulsive forces are minimized, electrostatic attractiveforces are maximized, and hydrophobic and hydrogen bonding forces aremaximized. Ligands bind to MBP in a specific and saturable manner, andbinding affinities may be measured according to ligand binding assaysknown to those skilled in the art, e.g. as disclosed further below.

"Ligand binding" between a murein binding polypeptide (or MB peptide)and a murein-, or murein-like, ligand is specific and saturable, and maye.g. be determined by incubating labeled ligand (e.g., radiolabeled orbiotin-labeled) at a concentration of about 0.1 fM to 10 mM (e.g., atroom temperature, 37° C., or 4° C.) with an aliquot of the MBP at aconcentration of about 10⁻⁴ to 10⁻¹² M. At lower binding affinities(e.g., 10⁻³ to 10⁻⁵), "bound" ligand is separated from "free", (e.g., bycentrifugation, filtration, column chromatography and the like) and theamount of labeled murein ligand is determined (e.g., by quantifyingradioactivity or reacting the sample with enzymatically-labeled avidin,washing to remove unbound avidin, and then adding substrate to visualizethe enzyme-bound-avidin-biotin receptor complex). At binding affinitiesgreater than 10⁻¹⁰ it may be difficult to measure binding by solid phasetechniques since binding becomes relative irreversible, but alternativemeasurement methods such as equilibrium dialysis, or sucrose densitygradient ultracentrifugation, i.e., using CsCl banded and sized cellwall fragments. Data obtained in these binding assays may be used toconstruct a Scatchard binding curve allowing determination of anassociation constant (Ka) that reflects the relative binding affinity ofa MBP for a ligand. Preferably, the subject MBP have a binding affinityfor diose, triose and tetraose bacterial and fungal cell wall compounds(e.g., chitatriose, (NAc-glucosamine)₃, and the like) that is in therange of about 10⁻³ L/mol to about 10⁻⁵ L/mol; and, the subjectcompounds are effective to bind the subject murein or murein-likecompound with a association constant (K_(a)) in the range of about5×10⁻⁵ L/mol to about 10⁻⁹ L/mol, preferably 5×10⁻⁷ L/mol to 10⁻⁹ L/mol,and most preferably greater than 10⁻⁹ L/mol. The binding affinity ofradiolabeled catalytically disable lysozyme to bacterial mureinscovalently bound to CNBr-Sepharose-4B (Pharmacia) was greater than about10⁻¹⁰ L/mol, i.e., the radiolabled MBP was not easily removed from theresin.

"Catalytically inactive" murein binding polypeptide (or peptide) isintended to mean that the subject MBP is capable of binding a mureincompound or a murein-like compound and it either completely lacks theability to catalyze cleavage of any bond in the subject murein compound,or it has only about 5% to about 30% of the activity of a correspondingwild-type enzyme, preferably only about 10% to about 15% of wild-typeenzyme activity, and most preferably less than about 10% of wild-typeenzyme activity. Most preferably, the subject catalytically inactive MBPexhibits a catalytic turnover rate of less than about 0.5 mmol to about3 mmole of the subject murein compound per mole enzyme per minute,preferably less than about 1 mmol to about 1.5 mmol substrate per moleenzyme, and most preferably less than about 1 mmol substrate cleaved permol MB polypeptide per minute. Representative examples of catalyticallyinactive murein binding polypeptides are disclosed in EXAMPLE 1, below,and include at least recombinant, mutant, chemically modified, andchemically inactivated polypeptides that have murein binding capabilitybut are catalytically inactive (as defined supra.) Examples includeenzymes having catalytic active sites with one or more amino acidresidues modified, inactivated, swapped/exchanged, mutated, chemicallymodified, derivatized, cross-linked and the like. Preferably, thesubject catalytic site amino acid residues, so modified, are those whichare capable in a wild-type enzyme of participating in a catalyticexchange mechanism. Illustrative assays for determining catalyticactivity are disclosed below (e.g., EXAMPLE 1.) In alternativeembodiments, ISBES methods (supra) are provided for producing killed anddenatured bacteria and fungal particles having murein ligands that mayconstitute good ligands but poor substrates. The subject ligands areproperly reactive with certain wild-type enzymes, i.e., lysozyme andchitinase, so that these wild-type enzymes may be used according to themethods of the ISBES methods described supra and illustrated in theExamples section, below.

"Non-interfering amino acid", as used herein, is intended to mean anyamino acid that when introduced into an MBP does not interfere withbinding of a murein ligand, but is effective to render the subjectenzyme catalytically inactive. For example, non-interfering amino acidsmay be useful to alter the spacing between adjacent or distant chemicalresidues, or to change the electrostatic charge distribution, orhydrophobic properties of an enzyme catalytic active site.Non-interfering amino acids may also be useful for terminal extension(N-terminal or C-terminal) of MB peptides to stabilize the subjectpeptides. Disclosed below are methods for altering enzyme active sites(e.g., by site directed mutagenesis, chemical modification, and thelike; EXAMPLES 1 and 3) and to render them catalytically inactive andsuitable for use according to the instant invention.

"MB polypeptide fragments" is used to mean those portions of an MBP thatare smaller in size than a wild-type MBP, i.e., isolated from a naturalsource (supra). Fragments may be prepared from a substantially purifiedMBP (i.e., preferably greater than 90%, and most preferably greater than95% pure, by SDS-PAGE) by proteolytic degradation (e.g., with trypsin,chymotrypsin, pronase, papain, subtilisin, and the like), oralternatively, by treating the subject polypeptide with a chemicalhydrolyzing peptide bonds (e.g., cyanogen bromide.) In the latter case,the fragments of the MBP are also preferably purified substantiallybefore use in the instant diagnostic reagents and assays, e.g., byreverse-phase HPLC or ligand-affinity chromatography, e.g. on a resincontaining one or more covalently bound murein compounds. Alternatively,fragments of an MBP may be prepared by expressing e.g. in a recombinanthost cell, a portion of a nucleotide sequence of a wild type (or mutant)genomic or cDNA clone capable of expressing the subject MBP. Host cellsare e.g. produced by introducing a nucleotide sequence, e.g., DNA or RNAintroduced by transfection, transduction or infection using e.g. in anexpression plasmid (or vector). Recombinant host cells include bacterialcells, insect cells, yeast cells and mammalian cells. The subject hostcell manufactures the subject MBP fragment which may be purified priorto use. Following purification, the subject MBP fragment may be testedto confirm murein binding activity.

"Specificity", when used in the context of an assay according to anembodiment of the invention, is intended to mean that the subject assay,as performed according to the steps of the invention, is capable ofproperly identifying an "indicated" percentage of samples from within apanel of biological samples (e.g., a panel of 100 samples). The subjectpanel of samples all contain one or more murein analytes (e.g., positivecontrol samples contaminated with bacteria or fungi.) Preferably thesubject "indicated" specificity is greater than 85%, (e.g., the assay iscapable of indicating that more than 85 of the 100 samples contain oneor more murein analyte), and most preferably, the subject assay has anindicated specificity that is greater than 90%.

"Sensitivity", when used in the context of an assay according to anembodiment of the invention, is intended to mean that the subject assay,as performed according to the steps of the invention, is capable ofidentifying at an "indicated" percentage those samples which contain amurein analyte from within a panel of samples containing both positivecontrols (supra) and negative controls (i.e., lacking murein analyte.)Preferably the subject "indicated" sensitivity is greater than 85% andmost preferably greater than 90%. "Background", when used in the contextof an assay according to an embodiment of the invention, is intended tomean the uncertainty in a test result, (sometime expressed as apercentage of false-positive or false-negative test results or by ameasurement of a degree of confidence in a test result), occasioned bysubstances which may interfere with the proper performance of the assaywhen they are present in the assay. Representative examples ofsubstances which may so interfere (i.e., interfering substances,confounding substances, and the like) in the assay include materialspresent in biological samples such as di- and trisaccharide cell wallcomponents of bacteria and fungi, endogenous murein binding polypeptides(defined supra), inhibitors or substrates for signal generatingcompounds (e.g., enzyme inhibitors, free radical reactive compounds,endogenous peroxides and the like.)

MBP suitable for use in the instant invention may also be prepared bychemical modification of an enzyme (e.g., TABLE 1). For example, asubject enzyme may be treated with a chemical selected for its abilityto render the subject enzyme catalytically inactive while preservingmurein binding activity. Esterification and methylation of lysozyme arerepresentative examples of a chemical treatments that destroy catalyticactivity (according to the invention, supra) while preserving mureinbinding activity. Another representative example is chemicalcross-linking to form enzyme-dimers wherein the tertiary structure ofenzyme is rendered rigid and/or entry and exit of substrates from acatalytic site is obstructed. Representative examples of enzyme activesite inhibitors that may be useful in preparing MBP include Allosamidin(7) and the like.

"Substantially purified" is used herein to refer to a preparation thatcontains a MB polypeptide, a MB polypeptide fragment, or a MB peptidethat is enriched greater than about 10-fold to about 25-fold, preferablygreater than about 26-fold to about 50-fold and most preferably greaterthan about 100-fold from the levels present in a source material. Thesubject preparation also preferably contains less than about 10%impurities, and most preferably less than about 5% impurities detectablee.g. by either SDS-PAGE or reverse-phase HPLC.

An MB peptide may be synthesized by any of a number of automatedtechniques that are now commonly available. Generally speaking, thesetechniques involve stepwise synthesis by successive additions of aminoacids to produce progressively larger molecules. The amino acids arelinked together by condensation between the carboxyl group of one aminoacid and the amino group of another amino acid to form a peptide bond.To control these reactions, it is necessary to block the amino group ofone amino acid and the carboxyl group of the other. The blocking groupsshould be selected for easy removal without adversely affecting thepeptides, i.e., by racemization or by hydrolysis of the formed peptidebonds. Amino acids with carboxyl-groups (e.g., Asp, Glu) orhydroxyl-groups (e.g., Ser, homoserine, and tyrosine) also requireblocking prior to condensation. A wide variety of procedures exist forsynthesis of MB peptides, solid-phase synthesis usually being preferred.In this procedure an amino acid is bound to a resin particle, and the MBpeptide generated in a stepwise manner by successive additions ofprotected amino acids to the growing chain. Modifications of thetechnique described by Merrifield are commonly used (Merrifield, R. B.,J. Am. Chem. Soc., 96: 2989-2993, 1964.) In an exemplary automatedsolid-phase method, peptides are synthesized by loading thecarboxy-terminal amino acid onto an organic linker (e.g., PAM,4-oxymethyl phenylacetamidomethyl) covalently attached to an insolublepolystyrene resin that is cross-linked with divinyl benzene. Blockingwith t-Boc is used to protect the terminal amine, and O-benzyl groupsare used to block hydroxyl- and carboxyl-groups. Synthesis is preferablyaccomplished in an automated peptide synthesizer (Applied Biosystems,Foster City, Calif.). Following synthesis, the product may be removedfrom the resin and blocking groups removed using hydrofluoric acid ortrifluoromethyl sulfonic acid according to established methods (Bergot,B. J. and S. N. McCurdy, Applied Biosystems Bulletin, 1987.) A routinesynthesis can produce 0.5 mmole of MB peptide-resin. Yield followingcleavage and purification is approximately 60 to 70%. Purification ofthe product MB peptide is accomplished for example by (i) crystallizingthe MB peptide from an organic solvent such as methyl-butyl ether,followed by dissolving in distilled water, and dialysis (if greater thanabout 500 molecular weight); or, by (ii) reverse HPLC (e.g., using a C18column with 0.1% trifluoroacetic acid and acetonitrile as solvents) ifless than 500 molecular weight. Purified MB peptide is commonlylyophilized and stored in a dry state until use.

Knowledge of the amino acid sequence of a MB polypeptide (or MB peptide)(i.e., the sequence of amino acids and spatial distribution of aminoacids in a sequence motif involved in murein binding permitsconstruction of recombinant host cell expression systems (supra) whereina cDNA (or genomic DNA) capable of encoding a wild type parental MBP(e.g., an enzyme) is modified (e.g., chemically or by site-directedmutagenesis) to produce a nucleotide sequence capable of encoding arecombinant catalytically inactive MBP. A representative example of thesubject modification of a cDNA is disclosed in EXAMPLE 1, below. Forsite-directed mutagenesis complementary oligonucleotides may besynthesized, or restriction fragments may be produced and chemicallymodified, in either case, the methods are available in the art.Incorporation of modified cDNAs (or gDNAs) into bacteria, yeast, andinsect plasmid DNAs, as well as into mammalian cell viral vectors (e.g.,retroviral vectors) may also be accomplished using techniques availablein the art. Host cell expression systems that may be useful forproducing MBP compounds include at least prokaryotic, eukaryotic, yeast,and insect cells. In one presently preferred embodiment, the cellularexpression system contains a tandem repeat from a sequence capable ofcoding for the subject MBP and 1 mole of the expressed protein productof the coding sequence is cleavable by cyanogen to yield about 2 mole ofthe subject MBP protein.

The present invention also provides that MBP may also be produced bychemical means, e.g., by derivatizing and covalently modifying awild-type MBP (e.g., an enzyme selected from Table 1), thereby renderingthe subject enzyme catalytically inactive and suitable for use in an MBPdiagnostic reagent. For example, modification of an MBP may include: (a)covalently modification, e.g. by adenylation, methylation,esterification, acylation, acetylation, phosphorylation, uridylation,fatty-acylation, glycosylation, and the like; (b) stereoisomerization,e.g., replacing a D-amino acid with an L-stereoisomer e.g. during solidphase synthesis; (c) derivatization, wherein one amino acid issubstituted for another of like properties by a series of chemicalmodifications or during solid phase synthesis, i.e., substitution of oneneutral polar amino acid for another neutral polar amino acid (e.g., G,A, V, I, L, F, P, or M); or, substitution of a neutral nonpolar aminoacid for another neutral nonpolar amino acid (e.g., S, T, Y, W, N, Q, orC); or, substitution of an acidic amino acid for another acid (e.g., Dor E), or a basic amino acid for another (e.g., K, R, or H); or, (d)chemically modification, e.g., converting an active site carboxyl groupto a carbonyl or aldehyde, or converting an amine to an amide, orintroducing a side chain at an active site residue e.g. Sar orgamma-amino butyric acid (GABA); or, (e) chemical coupling, e.g.,covalently coupling one active site residue to another residue usinge.g. a heterobifunctional cross-linking reagent (Pierce Chemical Co.);(f) or, chemical coupling to accomplish an N- or C-terminal extension(e.g. for an MB peptide); or, (g) replacing one amino acid with anotherof slightly different properties e.g., to change hydrophobicity of apeptide. In one presently preferred embodiment the MBP lysozyme wascatalytically inactivated by methylation or esterification.

Embodiments of the invention also provide increased binding affinitythrough use of multimeric MBP compounds and chimeric MBP compounds.Multimeric MBP compounds containing multiple copies of the same MBP maybe synthesized, or produced through genetic recombination, or chemicallycoupled the one to the other. Chimeric MBP compounds containing multiplecopies of two or more different MBP may combine advantageous propertiesof two different MBP molecules into a single molecule (e.g., combiningMBPs having two different anomeric or stereo specificities; or,combining an MBP having specificity for a murein oligopeptide side chainwith an MBP having specificity for sugar residues in a glycosyl chain);or, to increase the binding affinity of the subject MB-polypeptides to aligand through cooperative binding interaction between the respectivedifferent respective binding sites within the resultant chimericmolecule. In one representative example, lysozyme is coupled to achitinase to increase MBP binding affinity and broaden ligand bindingspecificity, as well as, to improve biochemical properties such ashydrophobicity and/or thermal stability. In another representativeexample, a bifunctional murein binding polypeptide consists of a portionof a lysozyme covalently coupled to a portion of a transglycosylase. Inyet another representative example, a bifunctional murein bindingpolypeptide consists of an enzyme (e.g., lysozyme) coupled to a mureinbinding lectin (e.g., neolectin.) In another representative example abifunctional murein binding polypeptide consists of an enzyme (e.g., anN-Acetyl-gluanase) coupled to a murein binding antibiotic (e.g.,vancomycin.)

Embodiments of the invention provide diagnostic reagents useful in assayformats for identifying bacteria and fungi and their cell wall productsin a variety of different types of biological samples. Representativeassay formats useful for detecting mureins include enzyme-linkedmurein-binding solid-phase absorbent assays (ELMBSA), radiolabeledmurein-binding assays (RMBA), fluorescence murein-binding assays (FMBA),time-resolved MB fluorescence assays (TRMBF), as well as, sandwich- andenzyme-cascade assay formats. Illustrative methods, as may be adaptablefrom the immunoassay art for use in the subject murein-binding assaysinclude: homogeneous assay formats; heterogeneous assay formats;competitive assay formats; non-competitive assay formats, enzyme-linkedsolid phase assay formats, fluorescence assay formats, time resolvedfluorescence assay formats, bioluminescent assay formats, and the like,examples of which are provided in the Appendix following the Examplessection, below. The instant murein-binding assay formats differ from theformer assays in their use of a non-antibody MBP, and the samplepretreatment and assay conditions necessary and effective to detect amurein compound (or murein-like compound) in a biological sample.Illustrative different MB assay formats are summarized in TABLE 4,below.

                                      TABLE 4                                     __________________________________________________________________________    Representative Murein Binding Assay Formats*                                                                 Detect Reagent                                                  Separation of Bound Murein                                                                  First  Second                                  Solid Phase                                                                            Capture Reagent                                                                       from Free     Partner                                                                              Partner                                 __________________________________________________________________________    Whole bacteria in                                                                      None    filter, centrifuge and/or wash                                                              MBP-SGC                                                                              None                                    test sample, filter or                                                                 None    filter, centrifuge and/or wash                                                              MBP#1  BP#1                                    c'fuge to collect                                                                      None    filter, centrifuge and/or wash                                                              None   BP#1                                    Plastic: polystyrene,                                                                  MBP#1   filter, centrifuge and/or wash                                                              MBP#2-SGC                                                                            None                                    PVDF, nylon 6.6,                                                                       MBP#2   filter, centrifuge and/or wash                                                              MBP#1  BP#1-SGC                                plates, filters, beads                                                                 MBP#1   filter, centrifuge and/or wash                                                              BP#1-SGC                                                                             None                                    Glass: amidated                                                                        MBP#1   filter, centrifuge and/or wash                                                              MBP#3  BP#3-SGC                                         MBP#3   filter, centrifuge and/or wash                                                              BP#3   None                                    Magnetic particles                                                                     MBP#1   filter, centrifuge, 1xg settle, pass                                                        MBP-SGC                                                                              None                                                     over magnet, and/or wash                                     Dipstick MBP#1 region on                                                      occurs during movement of                                                              MBP-SGC None                                                                  dipstick                                                                              mobile phase along dipstick                                  Dipstick MBP#1 region on                                                      occurs during movement of                                                              MBP-SGC None                                                                  dipstick                                                                              mobile phase along dipstick                                                                 bound to latex                                                                beads mobile                                                                  on dipstick                                    Dipstick MBP#1-SGC on                                                                          occurs during movement of                                                                   None   None                                             dipstick                                                                              mobile phase along dipstick                                                                 (e.g., UV                                               (e.g. SGC =           fluorescence                                            fluorophore);         quench by                                               MBP#2-latex           binding of                                              bead bound in         bead-MBP-                                               mobile phase)         analyte to                                                                    MBP#1-SGC)                                     __________________________________________________________________________     *SGC, signal generating compound; MBP, murein binding polypeptide; First      partner, first binding partner; Second partner, second binding partner;       MBP#1, specificity #1 of binding to a murein compound (e.g., specificity      for 1,4 linked glycosyl compounds); BP#1, specificity for binding to MBP#     (e.g., an antibiotic binding an MBP such as vancomycin); MBP#2,  #            specificity #2 of binding to a murein compound (e.g., specificity for         Nacetyl glucosamine); BP#2, specificity of binding to MBP#2; MBP#3,           specificity #3 of binding to a murein compound (e.g., specificity for         alanyl containing peptides); BP#3, specificity for binding MBP#3 (e.g.,       monoclonal antibody to MBP#3).                                           

The instant diagnostic assay formats and diagnostic reagents includethose useful for detecting endogenous and exogenous murein bindingpolypeptides at they are found in situ after binding to murein compoundsin the cell walls of eubacteria and fungi. "Endogenous murein bindingpolypeptides" is intended to mean MBP that are synthesized by bacteriaand fungi and become incorporated into the cell wall of these organisms.Representative examples of endogenous murein binding polypeptidesinclude bacterial peptidyl transferases which covalently crosslinkpeptidoglycan chains, and themselves become covalently bound in thebacterial cell wall. "Exogenous MBP" is intended to mean MBP that areadded to the bacteria or fungi; where they become bound to mureincompounds in the cell wall; then subsequently, they are detectablethrough use of an antibiotic-SGC conjugate that specifically binds tothe subject MBP. Embodiments of the invention provide reagents andmethods for detecting murein binding polypeptides in situ, i.e., as theyare bound in the cell walls of bacteria and fungi. Preferably, adiagnostic reagent for identifying a murein binding polypeptide in situincludes an antibiotic compound chemically conjugated to a signalgenerating compound, i.e., an antibiotic-SGC. An illustrative example isprovided in the Examples section below, wherein ampicillin waschemically bonded through its free amino group to NHS-fluorescein and isuseful in detect endogenous MBP in bacterial cells, i.e., endogenouspeptidyl transferases.

Representative antibiotic compounds that may be conjugated to a signalgenerating compound for detecting an endogenous or exogenous mureinbinding protein in situ in a bacterial or fungal cell wall, includeantibiotic selected from among the groups of penicillins, ampicillins,amoxycillins, vancomycins, streptomycins, erythromycins, bacitracins,tetracyclines, polymyxins, novobiocins, colistins, kanamycins,neomycins, ristocetins, gramicidins, spiramycins, cephalosporins,capreomycins, rifamycins and gentamycins.

The instant murein-binding assay methods include those having a stepeffective to simultaneously accomplish binding and signal generation asan analyte binds to an MBP, i.e., a "simultaneous" or "homogeneous"assay format. The instant methods also include "heterogeneous"murein-binding assay formats, including one or more steps for separatinga "bound" from a free analyte and then generating a signal. The instantmethods also include those having a step in which analyte is added tocompete with the binding of a labeled ligand to an MBP, i.e., acompetitive binding (or indirect) assay format, or alternatively, inwhich binding of an analyte to a MBP is detected by adding a second MBPhaving signal generating compound, i.e., a non-competitive (or direct)assay format. Illustrative methods for separating "bound" analyte,ligand or MBP from "free" include filtration, and column separation,magnetic separation, as well as attaching one or more of the reactantsto a solid phase. Illustrative murein-binding assay methods fordetecting a signal generating compound commonly include: using an enzymeas a SGC that converts a substrate to a visually orspectrophotometrically identifiable product), or alternatively, excitinga fluorescent SGC coupled to an MBP so that a detectable signal isemitted, e.g. at a different wavelength, e.g. fluorimetric analysis.

Commonly, coating polystyrene (e.g., 96-well Dynatech-Immulon II, Nunc,or similar plates) with a murein binding polypeptide at a concentrationof about 1 mg/ml in a carbonate buffer at pH 8 for about 16 hoursresults in binding of about 20 to about 150 μg/well to the solid phase.

Embodiments of the invention provide flow cytometric assays using anMBP-SGC conjugate as a bacterial or fungal identification reagent. Inone presently preferred embodiment, the cytometric assay is afluorimetric assay and the SGC is a fluorophore. The instant flowcytometric assays may be conducted with or without size discrimination(e.g., based on light scatter measurements); with or without a seconddye indicator (e.g., propitium iodide, FITC, F-NHS, RITC, and the like);and, with or without quantification (e.g., counting the number ofparticles positive in the assay for binding of the subject mureinbinding polypeptide (peptide) conjugates. Additionally, size gating maybe adjusted to discriminate between bacterial and/or fungal analytes ina biological sample, and/or to reduce background. Particle counting maybe used to quantify the number analyte particles binding anMBP-conjugate. Quantification of the number of analyte particles in abiological sample may be used to determine the severity of an infectionin a host in need thereof, e.g., a human patient or an animal.

Embodiments of the invention include, murein-binding solid-phase assayformats having at least the following two steps: namely,

In a first step, a murein analyte present in a biological sample is`captured`, by binding to a MBP compound that is attachedelectrostatically (or covalently) to a solid phase; and,

In the second step, bound bacterial murein analyte is detected byreacting it with a MBP "detect reagent" having a signal generatingcompound.

Other steps in the instant murein-binding diagnostic assay formats mayinclude one or more steps for pretreating a fresh, frozen or storedbiological sample (e.g., stored at 4° C., -20° C. -70° C. and the like).The instant pretreatment step may involve adding (i.e., before step 1,above) an acid, a base, a mucosidase, a detergent (e.g., TWEEN-20),and/or a protease or DNAase, and the like. The preferred pretreatmentstep decreases the viscosity in a sample; or, to increases thesolubility of the sample (e.g. a lung lavage, sputum or urine sample);or, to denature endogenous lysozymes or chitinases that can constituteconfounding or interfering substances in an assay; or, to inactivatebacterial and fungal cell wall compounds that can constitute backgroundinterfering substances in an assay; or, to increase exposure andaccessibility of a murein- or murein-like compound in a biologicalsample for binding by an MBP. A preferred step in the ISBES assaymethods described supra, is effective to accomplish the subject decreasein viscosity and increase in viscosity, in addition to the other aspectsdescribed supra. In one preferred embodiment, fungal murein-likeanalytes in a urine sample are collected by centrifugation orultrafiltration; the urine sediment is washed and then treated with achemical base solution at about 25° C. to about 100 C., preferably about25° to about 65° C. and most preferably about 55° C. to about 65° C. Thesubject treatment is preferably conducted for about 2 minutes to about30 minutes, preferably about 10 minutes to about 30 minutes, and mostpreferably about 20 minutes to about 30 minutes. Preferably, the shorterincubation times, i.e., about 2 minutes to about 9 minutes, and/or lowertemperatures, i.e., less than 45° C., are conducted with about 2M toabout 5 M of a strong base solution, e.g. sodium hydroxide, potassiumhydroxide, ammonium hydroxide, barium hydroxide, calcium hydroxide,potassium carbonate, sodium carbonate, potassium acetate and sodiumbarbital and like. Preferably, the pH of the chemical base solution isgreater than about pH 9 and most preferably greater than about pH 10.Preferably, intermediate incubation times, i.e., about 10 minutes toabout 14 minutes, are conducted with about 0.2 M to about 3.5 Mconcentrations of a strong base, e.g., KOH, NaOH and the like.Preferably, the longer incubation times, i.e., about 15 minutes to about30 minutes, are conducted with about 0.2M to about 2M concentrations ofa strong base, e.g., KOH, NaOH and the like. In all cases, the subjecttreatment is capable of killing bacteria and fungi, hydrolyzing a testbacterial or fungal polypeptide (supra), and producing killed anddenatured bacterial and/or fungal particles containing ligands reactivewith the instant MBP compounds. Following alkaline treatment(s), asubject urine sediment sample is then optionally washed, neutralized(e.g., using an HCl solution), and/or optionally treated with one ormore of a protease, a glycosidase, a sialidase and/or a periodate. Mostpreferably, following alkaline treatment and/or any optional treatment,the subject urine sediment sample is treated with an acetylationsolution capable of effecting N-acetylation of sugar residues in thesample. Representative N-acetylation solutions so capable, includesolutions of pure acetic anhydride at final concentrations of about 0.8%(v/v) to about 5% (v/v), preferably about 1% (v/v) to about 4% (v/v),and most preferably about 2% (v/v) to about 5% (v/v); also, solutions ofacetyl chloride at about 2M to about 5M; and solutions of acetylationreagents capable of achieving like effects without destroying theintegrity of a killed and denatured yeast particle. The combined subjecttreatments were effective to produce single particle suspensions ofkilled and denatured fungal cells suitable for use withlysozyme-fluorophore conjugates wherein the presence and number of theparticles were determined by counting in a flourescence flow cytometer.Other features of the instant murein-binding assay formats are disclosedbelow.

"Uniform assay format" is intended to mean that the molecule capable ofcapturing (e.g., MBP #1) a bacterial murein, e.g., on a solid phase, andthe molecule capable of detecting the captured bacterial murein (i.e.,MBP #2) are the same non-antibody MBP, e.g., MBP#l and MBP#2 (TABLE 4,above, are both lysozyme.) It is intended within this definition thatdifferent forms of the non-antibody MBP (as defined by MBP#1, MBP#2, andMBP#3, supra) may be used for the capture reagent and the detectreagent, i.e., lysozyme polypeptide as MBP#1 and a lysozyme fragment-SGCas MBP#2.

"Mixed assay format" is intended to mean that in a first step of theassay the molecule capable of capturing a murein compound is differentthan the molecule capable of detecting the captured murein in a secondstep, e.g., non-antibody MBP in the first step, (e.g., MBP#1-3, TABLE 4)and a lectin- or antibiotic-MBP (i.e., capable of binding the respectiveMBP) in the second step (e.g., BP#1-3, TABLE 4.)

In other alternative embodiments the invention provides mixed assayformats in which a MBP is used as a `capture` (e.g., MBP#3; TABLE 4) andan antibody-conjugate specific for the MBP or for the captured mureincompound is used as a `detect` (e.g., BP#3, TABLE 4.) The capturereagent may alternatively consist of an antibody and the detect reagentan MBP-SGC conjugate. Polyclonal and monoclonal antibody reagentsspecific for a murein, or MBP, may be prepared by standard methods,including (if necessary) conjugating the subject murein to a carrier(e.g., KLH or BSA) to increase its immunogenicity.

In one presently most preferably mixed assay format embodimentantibodies are specifically reactive with an MBP, rather than with amurein compound.

Monoclonal antibodies to bacterial cell wall compounds disclosed byShockman in U.S. Pat. No. 4,596,769, (resulting from immunization ofmice with bacteria and bacterial cell wall fractions; column 4, lines39-42), were tested and not found useful in the instant assays becauseof high background binding to non-bacterial and fungal materials inbiological samples. In the latter case, background nonspecific bindingmay perhaps result from cross-reactivity (i.e., at the antigen bindingsite); or, binding of compounds to the Fc portion of the antibody (e.g.,the C1q complement collagen-motif binding site); or, nonspecific bindingto immunoglobulins involving e.g. electrostatic or hydrophobic forces.

In certain other embodiments, the invention provides diagnostic reagentscontaining MBP that are detect reagents. The instant detect reagentscontain one or more signal generating compounds conjugated to an MBP.Representative methods for covalently linking SGC to MBP include thoseusing hetero-bifunctional cross-linking reagents that are reactive withcarbonyl, aldehyde, carboxyl, amino, disulfide and thiol groups of aminoacids, e.g., carbodiimide, N-hydroxy succinimide,N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), maleimide,succinimidyl-pyridylthioproprionate, m-maleimidobenzoylN-hydroxysuccinimide ester, succinimidyl pyridylthiopropionate, and thelike. Methods for linking SGC fluorophores to MBP include, e.g.,encouraging electrostatic interactions of the subject MBP with thefluorophore by placing the MBP in a buffer having a pH below itsisoelectric point (e.g., pH 2.5-3). An illustrative method for linking afluorophore to MBP is disclosed (EXAMPLE 2, below.) Methods suitable forlinking phycobiliproteins to MBP are disclosed in Stryer et al. (U.S.Pat. No. 5,055,556).

The instant MBP-SGC conjugates (supra) are prepared for commercialdistribution as detect reagents, preferably by solubilizing them in oneor more buffer solutions and then dispensing them into reagent bottles,packages and the like; or, alternatively, by lyophilizing them anddispensing them as powders into reagent packages. The subject buffersolutions may include additives (e.g., stabilizers), emulsifiers (e.g.,detergents), and the like for preserving the activity of the instantMBP-conjugate during storage; or, for promoting the binding activity ofthe instant MBP for a murein ligand or analyte in a murein-binding assayof the invention. Examples of agents that may be used to promote thesubject binding interactions include additives that decrease total fluidvolume in an assay (e.g., polyethylene glycol, sucrose and the like);and, agents that promote interactions by provide an electrostaticsurface in solution (e.g., dextran, polystyrene beads, polyacrylatebeads, and the like.)

Embodiments of the invention provide assay formats including competitiveand non-competitive, direct and indirect, quantitative andnon-quantitative assays including ELMBSA, RMBA, FMBA, TRMBF, and cascadeassay formats (supra). "Cascade assay formats" is intended to meandetecting a murein (or murein-like) compound through a process: wherein,a first signal generating compound (i.e., SGC #1) produces a productthat can be utilized by a second SGC #2 to produce a product which e.g.can be utilized by a third SGC #3. The subject cascade of products fromSGC #1-3 results in amplification which results in a greater overallsignal that could be achieved by any single SGC.

As a first representative example of a murein binding assay according tothe instant invention, an ELMBSA is conducted using MBP-SGC as both a`detect reagent` (supra) and MBP in a "capture reagent". In the subjectMBP-SGC the SGC is an enzyme. Steps in a ELMBSA include: (i) coating thesurface of an assay plate, e.g., a 96 well Immulon-II™ microtiter plate(Dynatech, Inc.), with the MBP-capture reagent (supra); (ii) adding analiquot of a biological sample for a period of time sufficient toaccomplish binding between an analyte in the biological sample and theMBP `capture reagent`, i.e., about 2 to about 60 minutes, preferablyabout 2 to about 30 minutes, and most preferably about 2 to about 15minutes; (iii) washing to remove unbound materials in the biologicalsample from the wells in the microtiter plate; (vi) adding the MBP-SGC`detect reagent`, i.e., MBP-enzyme conjugate and incubating for a timesufficient to allow for binding of the MBP-SGC to any analyte bound tothe capture reagent; (v) washing to remove unbound MBP-SGC detectreagent; (vi) adding a substrate for the enzyme signal generatingcompound; and, then (vii) incubating with the substrate for a period oftime sufficient to convert the substrate to a product that is detectablein the assay, e.g., spectrophotometrically or visually.

In a second representative example of a murein-binding assay accordingto the instant invention, a quantitative simultaneous competitive ELMBSAincludes the steps of: (i) coating a solid phase with one or more mureincompounds (e.g. bacterial or fungal cell wall fragments); (ii) in aseparate container mixing a predetermined amount of an MBP-SGC conjugatewith an aliquot of a biological sample for a time sufficient toestablish a binding interaction between the MBP-SGC (i.e., an enzymeSGC) and any analyte that may be present in the sample; (iii) adding theanalyte/MBP-SGC mixture to the murein-coated solid phase; (iv)determining the amount of MBP-SGC conjugate available for binding to thesolid-phase murein coating by separating the analyte/MBP-SGC mixturefrom the solid phase, e.g. washing to remove unbound analyte-MBP-SGC;and then, (v) incubating the separated solid phase with an enzymesubstrate to detect the presence and/or amount of the MBP-SGC-enzymebound to the murein-solid phase. The signal generated in the subjectcompetitive assay is inversely related to the amount of analyte in thebiological sample.

In a third representative example of a murein binding assay according tothe invention, a murein-binding fluorescence histochemical assayincludes the steps of: (i) collecting and fixing a biological sample(e.g., a tissue sample); (ii) incubating the sample with MBP-SGC (i.e.,SGC is a fluorophore ); (iv) washing the sample to remove unboundMBP-SGC conjugate; and, (v) detecting the bound MBP-SGC conjugate byfluorescence microscopy.

In a fourth representative example of a murein-binding assay accordingto the invention, a murein-binding cytometric assay (i.e., acytofluorimetric assay), for diagnosing severity of fungal infection ina subject in need thereof commonly includes the steps of: (i) collectinga biological sample (e.g., a urine sample); and aliquoting the subjectsample for testing ; (ii) concentrating any bacterial or fungal analytesin the sample (i.e., by centrifugation, filtration and the like); (iii)treating the subject aliquot by means effective to simultaneously killany pathogens, to eliminate interfering or confounding enzymes orcompounds present in the sample, to disrupt any aggregated fungal cellclusters, chains, strands and the like, and to obtain a suspension ofsingle fungal cells (e.g., treating with a solution of about 2 M NaOHand then, optionally, with a solution of protease, e.g., trypsin,supra); (iv) incubating the treated aliquot with MBP-SGC conjugate(e.g., SGC consisting of a fluorophore); (v) separating bound MBP-SGCconjugate from free/unbound MBP-SGC (e.g., by centrifugation asdisclosed in EXAMPLE 5, below); and (vi) quantifying by cytofluorimetrythe number of fluorescent particles present in the treated aliquot ofthe biological sample thereby to determine the severity of the infectionin the patient.

Embodiments of the invention also provide kits useful for identifyingeubacteria and fungi in a biological sample, e.g. a patient sample, or asample of water or air. A representative kit contains the following:namely, one or more reagent packages at least one of which contains aMBP-conjugate; an assay buffer; an optional assay surface, e.g., a tray,a vessel, or dipstick; a set of instructions; and one or more optionalassay calibrators or reference compounds (e.g., a positive and negativecontrol). In one presently preferred embodiment, a kit contains reagentpackages containing: (i) one or more pre-treatment solutions forexposing murein compounds in a bacterial or fungal cell wall (e.g.,NaOH, protease, glycosidases or periodates (supra), and as illustratedin EXAMPLE 8, below); (ii) one or more reference calibrator solutions(e.g., 0.25% glutaraldehyde fixed single cell yeast suspensions, cellwall materials coated on latex beads (i.e., polystyrene andpolyvinyltoluene homopolymers, and copolymers of these compounds withstyrenes), and the like); (ii) one or more sensitivity enhancingsolutions (e.g., acetylation reagent to increase the binding activity ofa murein compound, e.g., EXAMPLE 8, below); (iv) one or more mureinbinding conjugates (e.g., MBP-FITC, MBP-enzyme, and the like); (v) oneore more assay buffers or wash buffers (e.g., assay buffer containingPEG and/or detergents that promote binding between MBP and a mureincompound); (vi) one or more periodate solutions for oxidizingcarbohydrates and increasing the number of groups available foracetylation; (vii) one or more blocking buffers for reducing nonspecificbackground (e.g., solutions containing BSA or milk proteins); and (viii)one or more solid phase reaction surfaces upon which, or in which, theassay may be conducted (e.g., microtiter plates, dipsticks, strips, andthe like.)

EXAMPLE 1 Preparations of Catalytically Inactive Murein BindingPolypeptides

Preparations #1:

Mutant Catalytically Inactive Avian Lysozymes: Malcolm, B. A. et al.disclose (45) putative roles of the respective residues residing withinthe active-site of chicken lysozyme (EC 3.2.1.17) using a technique ofsite directed mutagenesis in a yeast shuttle vector derived from pBR322and pJDB219, and also using two synthetic primers: one designed toeffect a G to A change at base 237 and a second to effect a G to Cconversion at 186. Two resultant mutant lysozyme enzymes wereidentified, i.e., one having Asp converted to Asn, i.e., D52N, and thesecond having Glu converted to Gln, i.e., E35Q. The D52N mutant enzymereportedly exhibited less than 5% of the wild-type catalytic activityagainst Micrococcus luteus cell wall substrate while retaining somebinding affinity for chitotriose (GlcNAc)₃. E35Q exhibited no measurableenzyme activity (i.e., less than 0.1%±0.1% of wild type) but also stillbound substrate. Dissociate constants for enzyme-chitotriose complexeswere reported: namely, 4.1 μM for D52N-chitotriose complexes and 13.4 μMfor E35Q-chitotriose. The two mutant lysozymes were reportedly expressedand secreted by yeast transformants at levels of about 5 mg/L andpurified by affinity absorption to, and high salt elution from,Micrococcus luteus cell walls, or alternatively, by affinitychromatography on chitin-coated Celite A and elution with a concavegradient from 1 liter of 0.15 M acetate buffer (pH 5.5) containing 0.5 MNaCl to 250 ml of 1 M acetic acid followed by isocratic elution with 1 Macetic acid according to methods described by Kuroki et al. (4.) Sincebinding to simple di- and trisaccharides is not indicative of binding tocell wall compounds, experiments were conducted to determine (i) whetherD52N, E35Q, or catalytically active lysozymes could bind bacterial cellwall fragments; (ii) whether substrates for any of the three lysozymeswere exposed at the surface of the cell wall of intact bacteria andavailable for binding either of these lysozymes; (iii) whether intactbacteria and/or their fragments, as present in biological samples,contained intact substrates available for binding to any of the threelysozymes; (iv) whether potentially infectious samples could be treatedto kill pathogens without destroying the ability of any bacteria to bindwith a lysozyme; (v) whether a conjugate could be prepared with any ofthe three lysozymes; (vi) whether sufficient conjugate could be bound tothe available bacterial cell wall materials in a biological sample toallow their detection in an assay; (vii) whether the binding between anyof the lysozymes and the substrate in a biological sample would be ofsufficient sensitivity or specificity to allow detection of bacteria ina biological sample; (viii) whether the lower limit of sensitivity forbinding between a lysozyme and a bacteria in a biological sample wouldbe sufficient to allow the use of a lysozyme as a diagnostic reagent;and, (ix) whether natural chitinase, lysozymes and mammalian, avian orplant materials would constitute substances capable of confounding orinterfering in an assay using a lysozyme reagent. The results of thesestudies were remarkably encouraging, and surprisingly lysozyme, whichhas relatively poor catalytic activity with chitin substrates, was foundto bind intact fungal cells. Certain of the results obtained in thesestudies, as relevant to illustrate various embodiments of the invention,are detailed in the EXAMPLES which follow. The results of theseexperiments show that lysozymes and catalytically inactive lysozymes andmurein binding proteins are useful in preparation of diagnosticreagents.

Lysozyme and the subject D52N and E35Q mutant catalytically inactivelysozymes, produced by site-directed mutagenesis methods, are useful inpreparation of diagnostics reagents, and catalytically inactivelysozymes constitute a presently preferred example of a murein bindingpolypeptide, as defined, supra and as possessing the requisitecatalytically inactivity of an MBP. Diagnostic reagents incorporatingthe subject MB polypeptides into capture and detect reagents, and thelike, according to the instant invention, are illustrated further below.

Malcolm et al. (45) is incorporated herein by reference for illustrativemethods of measuring catalytic inactivity and association constant,e.g., from a soluble complex with a fluorigenic triose substrate.Catalytic inactivity can also e.g., be determined by incubating asuspension of Micrococcus lysodeikticus (as a substrate) with a testsample of the lysozyme preparation in an assay volume of 2.6 ml andtaking optical density measurements at 450 nm using a 1 cm optical path."Inactive enzyme" shows a decrease in A₄₅₀ that is less than about 0.5units per minute per milligram test enzyme protein. Catalytic inactivitycan also e.g. be determined using an N-acetylglucosamine hexamersubstrate labeled with 2-aminopyridine, e.g., using a method accordingto Hase et al. (48.)

Preparations #2:

Mutant Catalytically Inactive Bacteriophage Lysozymes: Heinz andMatthews (46) disclose T4 lysozyme double mutant N68C/A93C in whichsurface residues Asn68 and Ala93 are replaced (using site-directedmutagenesis) with cysteine residues. The cysteines allow formation ofdimers through disulfide exchange. N68C/A93C T4-lysozyme dimers purifiedby molecular sieve chromatography are catalytically inactive and retainbinding activity for murein substrates.

Preparations #3A:

Chemically Inactivated Lysozymes #1: Hinge-bending motion is requiredfor catalytic activity at the lysozyme active site cleft and forcooperation between a substrate binding site in the "right hand" side ofthe cleft and a catalytic site in the "left hand" side of the cleft.Conformational restriction of lysozyme protein is accomplished usingglutaraldehyde to react with free amino groups on the surface of thepolypeptide and form intra- and inter-molecular crosslinks that freezethe conformation of the protein.

Murein binding polypeptide, i.e., hen egg white lysozyme (Sigma ChemicalCo., St. Louis, Mo.) is dissolved in about 2 ml of 0.1 M PBS, pH 6.8 toa final concentration of about 2-5 mg/ml. Glutaraldehyde is diluted in0.1 M PBS, pH 6.8 from a commercial stock solution of 25% (EastmanChemicals, Rochester, N.Y.) to a final concentration of 0.10% and 1 mlis added dropwise, with agitation to the 1 ml MBP solution. After 3hours at room temperature the reaction mixture is placed into dialysistubing and dialyzed against PBS, pH 6.8 overnight at 4° C. Remainingreactive glutaraldehyde sites are blocked by adding 0.5 ml of 1 M lysinein PBS, pH 6.8, and incubating for 30 minutes. Catalytically inactiveglutaraldehyde-cross-linked lysozyme is purified by affinitychromatography at 30-37° C. on Micrococcus lysodeikticus cell wallfragments bound to cyanogen bromide-activated Sepharose. Catalyticallyactive enzyme elutes in the wash and catalytically inactive enzymeremains bound. Bound inactive enzyme is eluted by incubation overnightat 4° C. in PBS, pH 3 containing 0.2 M N-acetylglucosamine and 2M NaCl.Inactive monomers, dimers and multimers are separated by molecular sievechromatography on Sephacryl S-200 in PBS, pH 7.4. Inactive monomer isuseful for preparations of conjugates with signal generating compounds,i.e., in `detect reagents`, while dimeric and multimeric forms areuseful as `capture reagents`. Alternatively, catalytically inactivelysozyme may be purified by ion-exchange and/or molecular sievechromatography.

Preparations #3B:

Chemically Inactivated Lysozymes #2: Active site charge distribution isalso important for catalytic activity, e.g. Asp-52. Ethyl esterificationwith triethyloxonioum tetrafluoroborate at pH 4.5, e.g., according toParsons et al. (47)is used to modify charge and catalytically inactivatehen egg white lysozyme, (i.e., to less than about 1% to 10% of thecatalytic activity of the wild type untreated control enzyme), whileretaining murein binding activity for the protein. Enzymaticallyinactive enzyme is separated by Bio-Rex 70 column chromatography similarto that described previously (47). Similarly, methylation was used tomodify charge and catalytically inactivate hen egg white lysozyme.

Preparations #3C:

Chemically Inactivated Lysozymes #3: Amidation of Asp and Glutamic acidresidues through esterification and ammonolysis according to Kuroki etal. (41e) is used to chemically inactivate hen egg white lysozyme whileretaining murein binding activity.

EXAMPLE 2 Murein Binding Polypeptide Signal Generating ConjugateEsterified F-NHS-Lysozyme

Conjugates consist of both a MBP and a signal generating compound (SGC;supra.) The following example discloses the preparation of a MBP-SGCconjugate, i.e., esterified-lysozyme-MBP (Preparation #3B, above)chemically linked to F-NHS as the SGC, according to the method disclosedbelow.

The following steps were conducted in the dark: namely,

Purified esterified-lysozyme(lyophilized powder), 10 mg, was dissolvedin 1 ml of 0.03 M bicarbonate buffer, pH 2.5-3.0 containing 0.15 M NaCl.

NHS-Fluorescein (F-NHS), 0.5 mg, was dissolved in 500 μl of DMSO and50-100 μl of the resulting stock solution was added dropwise, withmixing, to the esterified-lysozyme solution. The resulting conjugationsolution was placed on ice for 2 hours.

Unconjugated F-NHS was removed by chromatography on a PD-10 column(Pharmacia, Piscataway, N.J.) that was equilibrated with 0.01 Mphosphate buffered, pH 7.0-7.4, 0.15 M saline (PBS). The first 2.5 mlemerging from the column contains fluorescent peaks that were discarded(i.e. conjugated denatured aggregated polypeptides); the second 2.5 mlemerging from the column was collected (i.e., MBP-SGC conjugate); andremainder of the material on the column (i.e., free F-NHS) wasdiscarded.

Concentration of F-NHS-esterified-lysozyme conjugate was estimated bydetermining optical density at 280 nm (OD₂₈₀) assuming an extinctioncoefficient for the conjugate equal to 1.0. For use in a diagnosticreagent the concentration of the F-NHS-conjugate was adjusted to 400-500μg/ml with PBS, pH 7.0. Preferably the absorbance ratio of F-NHS atOD₅₉₀ to MBP at OD₂₈₀ was about 1:1, because at higher ratios there wasmore nonspecific precipitate in the conjugation reaction, and at lowerratios there was less efficient signal generation in diagnostic assays.

EXAMPLE 3 Murein Binding Polypeptide Conjugates with Signal GeneratingCompounds

Preparation #1: Horse Radish Peroxidase-Catalytically Inactive-LysozymeConjugate (HRP-MBP-Conjugate #1):

Murein binding protein, i.e., mutant lysozyme (EXAMPLE 1, Preparation#2, supra), was conjugated to horse radish peroxidase (Grade I,Boehringer Mannheim, Indianapolis, Ind.) to achieve a mole ratio ofHRP:MBP of about 2:1. Most proteins do not contain free sulfhydryls, butthis is not the case for the N68C/A93C T4-lysozyme mutant describedabove, with free Cys₆₈ and Cys₉₃. In addition, disulfide cross-linked ininter- or intramolecular bonds in polypeptides are often susceptible tomild reduction (i.e., using 1-20 mM dithiotreitol; DTT) creating one ormore reactive sulfhydryl residues, without loss of binding site affinityfor a ligand. In either case, lysozyme free sulfhydryl groups arereactive with MBS (m-Maleimidobenzoyl-N-hydroxysuccinimide ester)cross-linking agent. In one example, a reactive sulfhydryl in HRP isgenerated by reacting free amino groups with SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate). Mild reduction of SPDPremoves the pyridyl group exposing a reactive sulfhydryl. Conjugationbetween MBS-MBP and SPDP-HRP is accomplished by reducing (i.e., withDTT) then mixing the compounds under conditions favorable for disulfidebond formation.

MBS-MBP: m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; PierceChemical Co., Rockford, Ill.) is dissolved to a concentration of 70 mMin dioxane. A 50-fold molar excess of MBS is added with stirring to asolution of N68C/A93C T4-lysozyme, and the mixture is incubated for 1hour at 37° C. Excess reagent is removed by G-25 chromatography. Themodified MBP elutes in the void volume is ready for reaction withthiol-HRP enzyme (below).

SPDP-HRP: 3-(2-pyridyldithio)-propionic acid N-hydroxysuccinimide ester(Sigma, #P9398) contains a blocked thiol present as a 2-pyridyldisulfide group. The pyridyl is removable under conditions that leavenascent disulfide bonds intact. The resultant thiol-enzyme is thenreacted with MBS-modified MBP to generate the HRP-MBP conjugate.

10 mg HRP (250 nmoles) is dissolved in 0.5 ml PBS, pH 6.8 containing0.15 M NaCl and chromatographed on a G-25 column to remove low molecularweight contaminants that might react with SPDP. The enzyme elutes in thevoid volume and can then be collected by visual inspection of the columneffluent (this protein has a distinct brownish color.) A fifteen-foldmolar excess of SPDP was added dropwise to the enzyme solution withstirring. The reaction mixture was left at room temperature for 30minutes and excess SPDP reagent is then removed by gel-filtration onG-25 (as above). Modified HRP elutes in the void volume and is wellseparated from the excess SPDP. There should be no loss of enzymeactivity and the HRP-SPDP conjugate is stable for >2 weeks when storedat 4° C. as the 2-pyridyl disulfide derivative. The extent of couplingis determined by taking a 50-100 μl aliquot of SPDP-HRP, diluting to 1ml with distilled water and measuring the absorbance at 403 nm and 343nm against a distilled water blank. The amount of 2-pyridyl sulfonereleased from the test sample is used to quantify the extent ofconjugation. To this end, DTT is added to a final concentration of 25 mMand the absorbance at 343 nm is read again after about 5 minutes. Thedifference in absorbance before and after addition of DTT represents theamount of 2-pyridyl sulfone released and is stoichiometrically relatedto the total amount of 2-pyridyl disulfide bound to the HRP in the testsample. The extinction coefficient for the released 2-pyridyl sulfone is8.08×10⁻³ M-1 cm-L and the extinction coefficient for HRP is 2.5 at 403nm. From the data the degree of coupling of SPDP is calculated. Underthe conditions described (i.e., with a molar ratio of 15-20\SPDP mole:mole HRP) an average of about 2.3 SPDP molecules are bound to eachmolecule of HRP.

HRP-MBP Conjugate: Enzyme-SPDP, i.e., HRP-SPDP (above), is reduced togive the corresponding protein-thiol for reaction with MBS-MBP. Suitableconditions that allow reduction of the protein-2-pyridyl derivative,with reducing intramolecular disulfides, are achieved at acidic pH,i.e., in 0.01 M Tris-buffered, pH 3.0, 0.15 M saline (TBS, pH 3), asfollows. First, the SPDP-enzyme derivative is passed over a G-25 columnequilibrated in TBS, pH 3, and the void volume protein peak iscollected. Next, DTT is added to protein peak to a final concentrationof 25 mM. After treatment for 30-40 minutes at room temperature thethiolated protein is separated from the low molecular weight reactants(i.e., 2-pyridylsulfone) by gel filtration on G-25 equilibrated in PBS,pH 7.2. Finally, the purified HRP protein-thiol is added to the MBS-MBPderivative (above) at about a 1:1 mole ratio and the mixture is allowedto stand at room temperature for 30-40 minutes. 2-mercaptoethanol isadded to a final concentration of 10 mM. Purification of the HRP-MBPconjugates is accomplished on Sephacryl-S200, or on Fractogel TSK usingFPLC (Fast Protein Liquid Chromatography; Pharmacia).

Preparation #2: HRP-MBP Conjugate #2:

In two separate but parallel preparative procedures, exposed aminogroups on MBP, (i.e., E35Q catalytically inactive lysozyme from EXAMPLE1-Preparation #1 supra), and on horse radish peroxidase (HRP) aremaleimidated using 6-maleimido caproic acid N-hydroxylsuccinimide ester(MCS). Molecular sieve chromatography on Sephacryl-S200 (Pharmacia FineChemicals, NJ) is used to separate maleimidated-HRP or -MBP monomersfrom aggregates and multimers in each of the respective differentpreparations. Purified monomers in each preparation are independentlythiolated by brief treatment with N-acetyl homocysteine thiolactone(AHTL) to achieve mole ratios of about 1-2 thiol groups per mole MBP (orHRP). (Thiol mole ratios are determined using the fluorescaminefluorometric method.) Thiolated-HRP and thiolated-MBP products are eachindependently dialyzed against 0.2M acetic acid, lyophilized and storedat 4° C. until use. For coupling each of the lyophilized reagents isdissolved separately in an aliquot of nitrogen-saturated 0.1 M succinatebuffer, pH 6.0 containing 0.04% EDTA. Coupling of the thiolated-HRP tothe thiolated-MBP is accomplished under the conditions described inPreparation #2, above (i.e., HRP-MBP Conjugate, supra), by mixing thetwo preparations together at differing ratios of thiol-HRP to thiol-MBP(i.e., 1:1, 2:1, 3:1 and 5:1.) The HRP-MBP conjugate is purified bymolecular sieve chromatography on G-25 and then Sephacryl S-200 andfractions containing HRP:MBP different mole ratios are collectedseparately for testing. Testing includes both a determination of HRPenzyme activity and murein binding ability. For long term storage at 4°C. or -20° C., the MBP-HRP-conjugate was stabilized by addition of:ferrous sulfate to a final concentration of 10⁻⁴ M, BSA to a finalconcentration of 1%; and Tween-20 to a concentration of 0.05%.

Preparation #3: Alkaline Phosphatase-Catalytically Inactive LysozymeConjugate:

Alkaline phosphatase (Type VII, Sigma Chemical Co., St. Louis, Mo.) isdialyzed to 100 mM Tris-HCL buffer, pH 8.0. To convert Alkalinephosphatase hydroxyl groups to chemically reactive aldehydes that formSchiff bases with amines in MBP, (i.e., mutant lysozyme from EXAMPLE1-Preparation #1, above), 0.5 ml of the dialyzed Alkaline phosphataseenzyme is mixed with 500 μl of a periodate solution (214 mg sodiumperiodate in 10 ml potassium phosphate buffer, pH 8.0) and agitated for1 hour in the dark at room temperature. Periodate is removed bycentrifugation and the pH adjusted to 8.0 with potassium carbonate. MBP,2 mg in 200 μl, is added and the mixture incubated for 1 hr. at roomtemperature in the dark, after which time the pH is adjusted to 6.0 withformic acid. Excess free aldehydes are reduced by adding 300 μl sodiumcyanoborohydride (6 mg/ml in 500 mM sodium phosphate buffer, pH 6.0) tothe reaction mixture. The resultant Alkaline phosphatase-MBP conjugateis purified by dialysis overnight to TBS, pH 8 followed by G-25 andS-200 column chromatography in TBS, pH 8.0.

Preparation #4: F-NHS-NHS Conjugates:

Fluorescein-NHS (F-NHS, Pierce Chemical Co., St. Louis, Mo.) was foundto produce more stable conjugates with MBP than fluorescein (FITC), andwith less precipitate formed during the conjugation procedure. A 1-3%(w/v) stock solution of F-NHS was prepared in DMSO. 50 μl to 100 μl ofthis stock solution was added dropwise to 1 ml of a 10 mg/ml solution ofMBP protein in 50 mM bicarbonate buffer, pH 8.5, i.e., catalyticallydisabled or catalytically active lysozyme (supra) to achieve a finalF-NHS concentration of about 1.0 to 7 mg/ml. After storage in the darkon ice at 4° C. for 2 hours (or overnight), free F-NHS was removed fromconjugated F-NHS MBP by dialysis (i.e., to PBS) or molecular-sievechromatography on G-25M (i.e., in PBS, pH 7.0; Pharmacia, PiscatawayN.J.; "PD10" column). Protein concentration was determinedspectrophotometrically at 280 nm assuming a molar extinction coefficientof 1.0, and the final concentration was adjusted to about 0.4-0.5 mg/ml.

EXAMPLE 4 Preparation of MBP Solid Phases

Preparation #1: Lysozyme-Nylon 6 Solid Phase:

Nylon-6 plates (or filters) are soaked in a 3N HCl solution for about 1hour at 37° C. After rinsing with distilled water the amidated platesare thiolated using 0.05 M N-Acetyl homocysteine thiolactone (AHTL) in 1M imidazole (in a saturated nitrogen atmosphere) and incubating at roomtemperature overnight. Thiolated plates (or filters) are washed 4-6times with 0.2 M acetic acid or until no thiol is detected in the washby Elmens reaction. The plates are stored under nitrogen until use.

MBP is maleimidated to about 1-2 maleimide groups/mole MBP usingN-hydroxy succinimide ester (MCS), the product is purified by molecularsieve chromatography (Sephacryl S-200), lyophilized, and stored undernitrogen at 4° C. until use.

Thiolated Nylon-6 plates (or filters) are soaked in nitrogen saturated0.1 M succinate buffer, pH 6.0, containing 0.04% EDTA and covalentcoupling is achieved by adding maleimido-MBP (above) at about 2 mg/mland incubating at room temperature (under nitrogen) overnight. Afterwashing to remove unbound MBP, free sulfhydryl groups are blocked bytreating with 0.15 M NaCl containing N-ethyl maleimide for about 30-45minutes. The plates are prepared for use by washing with 0.15 M NaCluntil the OD₂₈₀ of the wash solution is less than about 0.03.

Preparation #2: Lysozyme-Nitrocellulose Solid Phase:

Nitrocellulose filters (0.45μ pore size; Schleicher & Schull, #BA85) arewashed in 10 mM Tris-HCL buffer, pH 8 containing 20 mM NaCl and 0.1%Triton X-100. Circles, i.e., "dots", having a diameter of about 3-4 mmare scribed onto the surface of the nitrocellulose using an indelibleink.

Murein binding polypeptide, in this case lysozyme (Sigma Chemical Co.),is dissolved in 62.5 mM Tris-HCL buffer, pH 6.8 containing 0.1% SDS, 20mM DTT and 5% BSA. For loading onto the nitrocellulose 10 μl of the MBPsolution is added to each of the dots on the filter, and adsorption isachieved by incubation at room temperature for a time sufficient toretain murein binding capacity but eliminate catalytic activity, i.e.,15-30 minutes. Unbound sites on the nitrocellulose are blocked bywashing with 10 mM Tris-HCl, pH 7.4 containing 0.9% saline and 5% BSA(Tris-saline-BSA). The filter is stored in a humidified chamber on topof a Tris-saline-BSA saturated Whatman (Grade 1) filter paper until use.Coupling in the presence of 0.1% SDS and 20 mM DTT results in loss ofenzyme activity but retention of murein binding activity.

Preparation #3: Coupling to Glass: Glass-arylamine derivatives ofsilanized glass were prepared and MBP, i.e., lysozyme from EXAMPLE 1,Preparation #3, is covalently bound according to the following method.

Fine controlled pore glass beads (approximately 0.3-1 mm in diameter;Corning) were washed with 5% nitric acid and silanized by addition of10% γ-aminopropyl-triethoxysilane in distilled water at pH 3.45.Silanized glass is reacted with p-nitrobenzoylchloride in chloroformcontaining 10% (v/v) triethylamine as a scavenger for hydrogen chloride.The resulting nitro group is reduced using 10% sodium dithionite indistilled water and the resulting p-aminobenzoylaminoalkyl derivatizedglass is diazotized with nitrous oxide generated in situ by HCl andsodium nitrite. After washing MBP is added in Tris-HCl, 0.15 M saline,pH 8-9. Approximately 50 μg of MBP may be coupled per 50 mg glass beads.For use in a solid phase ELMBPA (below), the MBP-conjugated glass beadsare filtered into the pores of a glass fiber filter (Whatman, GF/C) andnon-specifically reactive sites in the glass fiber filter are blocked bywashing with Tris-HCl, 0.15 M saline, pH 7.2 containing 5% BSA. Theresultant surface has the advantage of having a high density of MBPcapture reagent per unit area of glass bead impregnated filter.

Preparation #4. Covalent Coupling of MBP to Polystyrene ContainingBeads:

Polystyrene beads, e.g., latex beads, are available commercially in avariety of sizes (e.g., 0.01μ-7 mm) and with surface carboxyl-,carboxylate-modified- , sulfate-, amino-, and amidated groups availablefor coupling to a MBP. Covalent coupling increases the stability ofbound MBP and ease of handling.

Preparation #4A: For cytometry, polystyrene beads having a diameter ofabout 0.5μ are collected by centrifugation and washed with 10 mMphosphate buffered pH 7.0 saline (PBS) until the optical density of thedecanted PBS wash solution is less than 0.010 at 600 nm. Covalentcoupling is achieved by activating about 0.2 ml of packed beads forabout 30 min. in 25 ml of pentane-1,5-dial (Sigma Chemical Co., St.Louis, Mo., #6-5882) at a final concentration of 0.5% in distilledwater, and with regular agitation. Following activation beads werewashed with 10-15 ml distilled water and then PBS. Covalent coupling wasachieved by adding a 1 mg/ml solution of MBP, i.e., F-NHS-lysozyme(supra).

Preparation #4B: For mobile latex phase dipsticks, polystyrene beadshaving surface carboxyl-groups and a diameter of about 0.01-0.1μ arecollected by centrifugation, washed in PBS, pH 6.5. The beads areactivated using water-soluble carbodiimide (i.e., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrogen chloride, 10 mg/ml distilled water, pH4.5-4.8); washing after 30 min.; and coupling to amino groups in MBP isachieved by adding a solution of 1 mg/ml MBP, (i.e., HRP-lysozyme,supra) in PBS, pH 6.5.

EXAMPLE 5 Urine Diagnostic Assays

Uses of the subjects assays according to the invention include detectionof bacteria in a biological sample consisting of a sample of urine.

Sample Preparation: A normal human urine sample was collected, varyingnumbers of E. coli or Candida albicans cells were added to separateurine aliquots to simulate samples from an infected individual, and thento concentrate the bacterial or fungal particles the samples were eithercentrifuged 3000 rpm in a clinical centrifuge for 10 minutes, orfiltered through a Whatman glass fiber or Millipore 0.22μ pore filter.

For samples containing bacteria, the centrifugal pellet (or filtrate)was treated to kill infectious material, i.e., by fixing and killingwith 80% ethanol for 5 minutes or longer, or alternatively, by alkalinehydrolysis (as described below.) For samples containing fungi, thecentrifugal pellet (or filtrate) was subject to alkaline hydrolysis tokill and denature fungi, disaggregated fungal clumps, and removedconfusing and interfering substances (supra.) The conditions foralkaline treatment of the urine sediment sample were found to be crucialto obtaining uniform suspensions of killed and denatured particles, andin turn, for obtaining meaningful results in cytometric particlecounting assays. Urine sediment samples contained aggregated bacterialand fungal rafts, clusters and clumps, as well as, particulate matterthat bound bacteria and fungi and interfered with performingmeasurements in the assay. Alkaline hydrolysis and/or supplementaltreatment with one or more proteases, were evaluated includingtreatments with 0.2 M, 1 M, 1.5 M, 2.0 M, and 5 M NaOH at 37° C., 45°C., 60° C. or 100° C. for 5 minutes, 10 minutes or 30 minutes.Treatments with the lower concentrations of NaOH, and/or for the shortertimes, and/or at the lower temperatures killed and denatured all testbacteria and fungi, as determined microscopically by refractility andmicrobiologically by plating and culturing. Under the lesser denaturingconditions (e.g. 1 M NaOH for 2-10 minutes) some rafts and aggregates offungal cells were observed, and these could dispersed into singleparticle suspension by following the alkaline treatment with a proteasetreatment, i.e., 0.25% w/v trypsin for 2-15 minutes. Conditions givingexcessive killing and denaturation were adopted as a standard protocolto provide an extra margin of safety, i.e., treatment with 2 M NaOH at60° C. for 30 min. and because they eliminated aggregates. (The marginof safety necessary for different types of biological samples will, ofcourse vary.) The fixed and/or killed and denatured bacterial and fungalparticles were next collected (e.g., by centrifugation or filtration,supra) and resuspended e.g., in 200 μl PBS containing a finalconcentration of 2.5% BSA (PBS-BSA.)

Fluorescence Microscopy: Assay #1:

50 μl of the MBP-conjugate reagent catalytically inactive F-NHS-lysozyme(Preparation #1, supra) or catalytically active F-NHS-lysozyme (Sigma)was added the urine sediment or filtrate (supra) at a final proteinconcentration of about 40-80 μg/ml. The binding to bacterial murein orfungal murein-like compounds in the biological sample was accomplishedover about 10-30 minutes at room temperature.

"Bound" reagent F-NHS-lysozyme (i.e., bound to any bacterial or fungalparticles in the sample) was removed from "free" by centrifugation(above) and the particles were washed 2-3 times with about 100 μl eachof PBS-5% BSA, pH 7.0. The final sample was resuspended in about 10 μlof PBS-5% BSA, pH 7.0 and the presence (or amount) of F-NHS-lysozymebound to the particles in the sample was determined visually byfluorescence microscopy at a magnification of 1000×. Normal urinepurposefully contaminated with known numbers of either E. coli orCandida albicans gave positive test results (i.e., visible fluorescentparticles) in the assay.

Flow Cytometry: Assay #2:

Despite manufacturers claims to the contrary, bacteria and fungi arerelatively difficult to detect by flow cytometry because their sizefalls at the lower limits for detection by ordinary (non-laser) forwardlight scattering methods.

Bacteria or fungi are detected by adding 50 μl of F-NHS-lysozymecovalently bound to 0.5μ polystyrene beads (Solid Phase Preparation #4A,supra) to a treated urine sediment (prepared, supra). After incubatingfor about 10 min. to about 20 min. to effect binding of theF-NHS-lysozyme-beads to bacteria or fungi in the sample forming dimericand multimeric aggregates, the number of multimers is determined bycytometry as follows. First, to exclude counting of monomeric beads thelower detection limit of a cytometer (i.e., Coulter Instruments) is setto greater than 0.5μ using 0.5μ calibration beads. Next, measureddilutions of the incubation mixture containing bacteria or fungi boundin multimeric detection beads, is gently vortexed to create a uniformsuspension and then introduced into the flow cytometer. Multimers havingdiameters greater than 0.5μ are counted, and the number of suchparticles is an indication of the presence, or amount, of bacterialcontamination in a biological sample, or the severity of infection in ananimal.

Flow Fluorimetry: Assay #3:

Bacteria (E. coli) from in vitro culture, containing clumps andaggregates, were suspended in PBS, stained using F-NHS-lysozyme (200μg/ml/ 30 minutes/ 37° C.), and detected using fluorescence microscopy,or fluorescence flow cytometry in a Becton Dickinson FAXSCAN system(LYSIS II, ver. 1.1, 1992.)

FIG. 1A shows background auto-fluorescence in the E. coli sample priorto staining with F-NHS-lysozyme.

FIG. 1B shows F-NHS-stained bacterial aggregates (shaded peak) separatedfrom the superimposed peak (unshaded peak) of auto-fluorescence shown inFIG. 1A.

The numeric data recorded by the flow cytometer, i.e., correspondingwith FIGS. 1A and 1B, is presented in TABLE 5.

                                      TABLE 5                                     __________________________________________________________________________    Cytometry Data: E. coli Urine Sediment Sample                                         Window.sup.a                                                                        Particles                                                                          %  Peak.sup.b                                                                        Peak Mean Median                                    Figure                                                                            Channel                                                                           L  R  Counted                                                                            Total                                                                            Counted                                                                           Channel 1                                                                          Intensity                                                                          Intensity                                                                          S.D.                                 __________________________________________________________________________    1A  0   1.00                                                                             9646                                                                             10,000                                                                             100                                                                              705  1    6   3.8   15                                  1A  1   19.8                                                                             9646                                                                               286                                                                               3      21   42   28   80                                  1B  0   1.00                                                                             9646                                                                             10,000                                                                             100                                                                              204 111  182  120  256                                  1B  1   19.8                                                                             9646                                                                               9548                                                                              96                                                                              204 114  190  129  259                                  __________________________________________________________________________     .sup.a Window, fluorescence intensity settings for lower and upper limits     .sup.b Peak Counted, number of particles counted at the peak of               fluorescence intensity.                                                  

Flow Fluorimetry: Assay #4:

Endogenous murein binding polypeptides in bacterial cell wall weredetected using an antibiotic-SGC conjugate, i.e., ampicillin chemicallybonded through its free amino group to F-NHS. Briefly, ampicillin wasmixed with F-NHS (Pierce Chemical Co.) according to methods describedsupra in regards to preparation of F-NHS-lysozyme. Since only one freeamino group was available for binding, the stoicheometry of binding was1:1. Antibiotic-SGC conjugate was separated from free F-NHS and freeantibiotic on a P2 sizing column, i.e., the conjugate was about twicethe size of the free antibiotic. The F-NHS-ampicillin was used to stainendogenous peptidyl transferases in the cell walls of bacteria (i.e., E.coli) and fungi (i.e., Candida albicans) and stained bacterial andfungal cells were detected using flow cytofluorimetry and microscopy.Because of its small molecular size F-NHS-ampicillin was able to stainviable bacteria and fungi, as well as killed and denatured bacteria andfungi.

EXAMPLE 6 Enzyme Linked Murein Binding Assay ELMBA Solid Phase SandwichAssay

Murein binding polypeptide (MBP) capture reagent, in this casecatalytically inactive hen egg white lysozyme from EXAMPLE 1 (above), isdissolved immediately before use in 0.05 M sodium carbonate buffer, pH9.6 to achieve a final concentration of about 100 μg/ml. A solid phase(supra) is coated with MBP by adding 100 μl of the MBP-capture carbonatesolution to each well in a 96 well microtiter plate (ELISA standardpolystyrene; Nunc Inc. or Immulon-II, Dynatech Inc.). After coating theplates at 4° C. for 16-20 hours, unbound MBP is removed by washing 4-6times with 0.01M sodium phosphate buffer (PBS), pH 7.0 containing 0.5%Tween 20 (PBS/Tween). Unbound sites on the polystyrene are blocked byadding PBS, pH 7.0 containing 10 mg/ml of bovine serum albumin (BSA) and0.5% Tween-20 (Blocking Buffer), and incubating for 3-4 hours at roomtemperature. Blocking buffer is removed by washing with PBS/Tween andthe microtiter plates are covered and stored at 4° C. until use. Testsamples of biological fluid, i.e., a urine sediment sample (supra), areprepared as serial dilutions in PBS, pH 7.0 and 100 μl of each dilutionis added to a well of the coated microtiter plate containing the boundMBP. Binding between the MBP and any bacterial murein, or fungalmurein-like, analyte in the test sample is allowed to continue for 45minutes to 1 hour at room temperature, and then unbound materials areremoved by washing 4-6 times with PBS/Tween. Bound murein, ormurein-like, analyte is detected stepwise: first, by adding 100 μl/wellof a detect reagent HRP-lysozyme-conjugate (EXAMPLE 3, above) dissolvedin PBS, pH 7.0. The reaction between the detect reagent and thebound-analyte is allowed to continue at room temperature for 30-90minutes and unbound detect reagent is next removed by washing withPBS/Tween, followed by PBS. Second, the bound detect reagent isvisualized by adding 100μl of a colorimetric substrate for HRP, i.e., asolution of ABTS (2,2"-azino-di(3-ethylbenzthiazoline sulfonic acid) ata concentration of 0.16 mg/ml in PBS, pH 6.0 and containing 0.0004%(freshly prepared) hydrogen peroxide. Visible color development isallowed to continue for 10-60 minutes, and absorbance is quantifiedspectrophotometrically at 405 nm (i.e., in an ELISA plate reader).Third, the results are quantified by comparing the absorbance recordedwith the absorbance produced by measured amounts of murein ligandstandards run in parallel, i.e., in other wells of the MBP-capturemicrotiter plate during the assay of the test substance. The absorbanceproduced by the standards is used to construct a standard curve, and theabsorbance value for the test sample may be compared with the standardcurve to determine the bacterial-equivalents of a murein, ormurein-like, ligand in the sample. The results, may be used to setacceptable cut-off values of reactivity for "normal" in the assay, e.g.,an "infection" may indicated if a value obtained with a test sample froman individual is more than one standard deviations greater than a meanbackground value recorded with a panel of sample from normal healthyindividuals. Individual cut-off values may also be indicated fordifferent clinical grades (severity's) of infection.

EXAMPLE 7 Dot-blot Filter Assay for Biological Samples

Assay #1:

A solid phase filter is coated with an MBP capture reagent, in this casea Whatman glass fiber filter (GF/C) impregnated with fine controlledpore glass beads to which lysozyme is covalently linked, i.e., EXAMPLE4, Preparation #3.

A sample of a biological fluid, i.e., a cerebrospinal fluid (CSF) sampleis collected in a sterile manner and 200 μl aliquots of neat(undiluted), and diluted samples are prepared using 10 mM Tris Buffered,pH 7.2 0.15 M saline (TBS). The aliquots are each subject to pressurefiltration (e.g., applied by a syringe) through a separate portion(i.e., a "dot") of the MBP-coated-filter (supra). Reactive sites on thefilter are blocked by washing "blocking buffer", i.e., TBS containing0.5% Tween and 1 mg/ml BSA, supra, through the filter. After washing thefilter with 5-10 ml blocking buffer, bacteria, fungi and/or cell walldegradative products trapped on the filter are visualized by adding 100μl of detect reagent, i.e., lysozyme-alkaline phosphatase conjugate(EXAMPLE 3, Preparation #3, supra.) After an additional 15-45 minutesincubation at room temperature, unbound detect reagent is removed bywashing with about 5-10 ml TBS. The presence or amount of bacteria orfungi in the biological sample is visualized by adding about 1 ml of analkaline phosphatase substrate, (i.e., 1.5 mM bromo-chloro-indolylphosphate in 1.0 M diethanolamine containing 0.5 mM magnesium chloride,pH 9.6), and incubating until color appears. Quantification of the boundbacteria or fungi is accomplished by eluting the blue product from thefilter in 200-500 μl of 80% ethanol, followed by spectrophotometricanalysis at 605 nm and comparison against standards.

Assay #2:

Conditions for this assay are as above (i.e., Assay #1), but the MBPdetect reagent is a lysozyme-HRP-conjugate, (i.e., EXAMPLE 3,Preparation #2.) Peroxidase substrate used in this assay is4-amino-antipyrene whose product precipitates from solution as a visiblered color on the glass fiber filter, and the substrate solution consistsof 0.2 mM 4 amino-antipyrene in 0.01 M sodium phosphate buffer, pH 7.5containing 0.0004% (fresh) hydrogen peroxide and 20 mM phenol.

EXAMPLE 8 Diagnostic Assay for Fungal Infections

The following example illustrates the instant diagnostic methods as usedto rapidly identify a bacterial or a fungal infection; or, to rapidlydifferentiate between a bacterial and a fungal infection in a host inneed thereof; or, to quantify the severity of a fungal infection in apatient in need thereof, e.g. a urinary tract infection.

Urinary Tract Infections:

Urinary tract infections (UTI) include urethritis (infection of theurethra), cystitis (bladder infection) and pyelonephritis (kidneyinfection.) Bacteria and fungi are common etiologic agents in UTI. Mostbacterial infections are E. coli, Klebsiella or Proteus and fungalinfections are yeast of the genus Candida (e.g., C. albicans, C. kruzei,C. glabrata, C. tropicalis and the like) and fungi of the genusTorulopsis (e.g., T. glabrata) and Trichosporon (e.g., T. beigelii.)There are great differences in the assay sensitivity required to detectbacterial or fungal infection. For instance, threshold for UTI forbacterial infection is considered to be greater than about 10⁵ colonyforming units (CFU), as contrasted with only about 1000 CFU/ml urine toconfirm a yeast infection. Most antibiotic treatment is initiatedbefore, or without, identification of the etiologic agent becauseseveral days are usually required to culture the bacteria or fungiresponsible. Widespread use of antibiotics has favored emergence ofantibiotic resistant bacteria and fungi, and the growing prevalence ofresistant microbes (particularly in the clinical environment) hascomplicated treatment of patients. Among patients with fever and pyuriaunresponsive to antibiotics the mortality rate is presently greater than30%. In addition, several patient groups are at an increased risk ofdeveloping life-threatening ascending infections capable of developinginto kidney and/or systemic infections: namely, immunocompromisedpatients (e.g., transplant, cancer patients, AIDS patients) includingpatients treated with immunosupppressive drugs (e.g., patients withautoimmune diseases such as IDDM, SLE, RA); indigent, elderly anddiabetic patients; and, hospitalized patients (e.g., nosocomialinfection, prostate disorders, pregnant women.) As a means for reducingcosts, managed healthcare will eventually mandate early diagnosis anduse of methods for distinguishing at an early stage between bacterialand fungal UTI infections.

Clinical Diagnostics Laboratory Testing:

Current methods of plate culture and microscopic examination are tooslow (i.e., days) to provide information useful in deciding treatment,and instead are used, (after the fact), to confirm bacterial, and/orfungal, infection. Current microscopic wet mount and/or plate culturemethods are used to identify yeast in urine sediments. For detection bymicroscopic examination, more than 1000 colony forming units of yeastper milliliter of urine may be required; and, for detection by culture,2 or more days may be required since yeast are relatively slow growing.As a result, false-negative test results and uncertainty are common.Alternative methods such as ELISA, calcofluor and PCR are expensive,slow, not easily automated, not pan-fungal reactive, often reactive withmammalian cell elements and bacteria, and have high backgroundnon-specific reactivity with non-fungal particulate matter (e.g., cottonlint). Performing these assays also commonly requires specially trainedpersonnel. Existing techniques also do not commonly aid the physician toidentify or determine a severity of infection or provide informationuseful in deciding whether to treat with an aggressive course ofhigh-dose antibiotic therapy.

Current Limitations to Flow Cytometry Methods

Flow cytometry has limitations when used for automated counting ofinfectious microorganisms including: high background counting ofnon-microbial particles; the small numbers of microorganisms present(e.g., <1000 cells/ml yeast) necessitating long counting times at thelower limits of machine counting. For a 10 ml urine sample, it ispreferable to count about 7,000-10,000 cells to diagnose a yeast UTI,but using e.g. a urine sediment sample of 500 μl, counting at 20 cellsper second at a flow rate of 60 μl/minute may require 6-7 minutes persample to record this number of cells. As a further complication forcytometric counting, it was discovered that bacteria and fungi in urinetend to aggregate and form into clumps, rafts and aggregates which maybe commingled with mammalian cell debris in the samples (FIG. 2A.)

FIG. 2A shows a fluorescence photomicrograph of staining of yeastclusters and aggregates observed when Candida albicans was added tonormal urine sediment and stained with F-NHS-catalytically disabledlysozyme.

Filamentous fungi also grow in chains or hyphae that are not conduciveto counting by cytometric methods. Due to these combined confoundingproperties, particle counting methods may often under-estimate theactual numbers of cells by more than 5-10 fold. Similarly, culturemethods may under-estimate the numbers of fungi by at least 2-3 foldbecause many yeast colonies originate from cell clusters (Perfect,1991.)

The Instant Assay Methods: Summary Overview

Since biological samples from infected individuals contain bacterial andfungal aggregates, as well as, `confounding` and `interfering`substances (supra), i.e., murein binding compounds such as endogenouschitinases and lysozymes attempts were made to produce single cellfungal and bacterial suspensions by combinations of acid and/or basehydrolysis and protease treatment.

As detect compounds, F-NHS-labeled catalytically-disabled lysozyme(i.e., Preparation #1 supra) and F-NHS-lysozyme and were preparedaccording to EXAMPLE 1 and comparisons were made of their effectivenessfor detection of alkaline and/or protease killed and denatured bacteriaand fungi by flow cytometry. Most pathogenic fungi contain chitin intheir cell walls, septa and spores, both in hyphal and yeast forms.Chitin is a β1→4 linked linear polymer of 2-deoxy-2-acetamidoglucose(N-acetyl glucosamine, GlcNAc.) Fungi do not contain bacterial mureinproteoglycans and chitin is a poor substrate for lysozymes withhydrolysis of chitins proceeding at a much slower rate than mureins.Similarly, bacteria do not contain chitin and are not reactive withchitinase.

Surprisingly, using fluorescence microscopy and Candida albicans as atest fungi, F-NHS-labeled catalytically-disabled lysozyme andF-NHS-lysozyme both stained yeast cells, and much more brightly than thestaining that could be achieved using a chitin-specific binding protein,i.e., chitin glycosyl hydrolase-F-NHS conjugate. Neither F-NHS-labeledconjugate stained mammalian or plant cells or their products. Alsosurprisingly, under the conditions of use, staining of bacteria in urinesediments was not as easily detectable as staining of fungi, i.e.,because of their size. In other words, under the conditions of use incytofluorimetric assays the method was able to selectively distinguishbetween the presence of bacteria and/or fungi in a test sample.Fluorescence labeling of Candida by both the F-NHS-catalyticallydisabled lysozyme and the F-NHS-lysozyme depended upon the preparativemethod (supra) used to prepare a biological sample for analysis.Fluorescence signal generation was markedly greater, i.e., as measuredby fluorescence intensity in fluorescence cytometry, when a test Candidayeast in a urine sediment was subject to alkaline denaturation followedby N-acetylation, but not O-acetylation. While not wishing to be tied toany particular mechanism of action, but by way of possible explanation,lysozyme binding specificity for yeast cells may require the presence ofmurein-like compounds and lysozymes may not effectively bind chitosansin a fungal cell wall with sufficient affinity to generate a signal inthe assay. Chitosan is present as a natural cell wall material in fungi,or as a possible result from deacetylation following alkaline treatmentprocedures (supra). Chitosan may be converted to murein-like compoundsby N-acetylation (supra.) In different experiments, the conditionssuitable for N-acetylation, without destroying the killed and denaturedyeast particles in urine sediments, were found to be treatments at afinal dilution of about 1/20-1/40 acetic anhydride (i.e., 5-10 μl for200 μl to 300 μl total sample volume; about 1-10% acetic anhydride finalconcentration); using chemically pure acetic anhydride (e.g., distilledto remove acetic acid); and, in a saturated solution of NaHCO₃ at pH 8.0for 15-30 minutes at room temperature. Using the combination of bothalkaline hydrolysis and N-acetylation the killed and denatured yeast orbacterial particles in the treated urine sediment were fully reactivewith the lysozyme F-NHS-conjugate (EXAMPLE 1, Preparation #1, supra). Inthe absence of an alkaline treatment the signal was dramaticallyreduced. Protease treatment did not increase the signal of a sampleexposed to both treatments, but it did increase the signal of a samplewith fungal aggregates and subject to only the alkaline treatment.However, periodate oxidation (i.e., increasing the number of free sugarresidues available for acetylation) followed by acetylation did increasesignal strength in certain samples.

In comparative studies, killed and denatured fungi in alkaline- andN-acetylation-treated urine samples were as reactive with F-NHS-lysozymeas with the catalytically disabled lysozyme conjugate (Preparation #1,EXAMPLE 1, above.) The mechanism responsible is at present unknown. Thetreatment method seems to produce killed and denatured fungal particlesthat contain substrates for catalytically active lysozyme conjugates,but, substrate turnover in the particles seems to be at a reduced rate,or turnover may not result in release of substantial free enzymefollowing the catalytic reaction.

Illustrative Urine Sediment Sample Treatment Protocol

One to ten milliliters of urine was centrifuged at 3000 rpm in aclinical centrifuge at room temperature (i.e., 22° C.) for 10 minutes.The urine sediment was resuspended in 100 μl 2N NaOH and subject tohydrolysis for about 15-30 minutes at 60° C. to kill bacteria and fungi;to hydrolyze and denature polypeptides and cell wall fragments; toexpose murein- and murein-like-compounds in the particles; to alkylatecell surface charged residues involved in aggregation; and, todisaggregate bacterial or fungal rafts, aggregates and cell clumpswithout lysing cells. Following alkaline treatment, the killed anddenatured particulate suspension was brought to neutrality by adding 30μl of 6N HCl and setting it aside at room temperature for 10 minutes. Ifaggregates, rafts or clumps of particles remained, the denaturedcellular proteins in the particles were subject to an optional step ofenzymatic hydrolysis, i.e., protease treatment with 0.25% trypsin for 30minutes at 37° C. in a volume of about 100 μl PBS, pH 7.0. The chemicaltreatment alone was usually sufficient to hydrolyze bacterial and fungalintracellular adhesins; degrade potentially confusing polypeptides(e.g., endogenous chitinases, lysozymes and the like); degrade mostpotentially interfering substances (e.g., mammalian cells, bacterialcells, bacterial cell wall fragments, fungal cell wall fragments and thelike); degrade fungal casts (e.g., releasing cells from hyphae); andrelease individual fungal cells from rafts, clumps, aggregates and thelike. To increase the reactivity of murein- and murein-like compoundswith MBP, i.e. lysozyme, the killed and denatured particles were subjectto N-acetylation by adding 100 μl saturated NaHCO₃, pH 8, followed by5-10 μl of pure (redistilled) acetic anhydride, i.e., finalconcentration in the assay of about 2-5% (v/v.) (N-acetylation was usedto convert chitosan and other fungal compounds into more murein-likecompounds reactive with MBP.) After 2-10 minutes at room temperature100μl of PBS, pH 7.0 was added, followed by 100 μl of PBS-5% BSA, pH7.0, i.e., bringing the total volume to about 400-500 μl. The particleswere detected by adding 30-40 μl of a solution of F-NHS-lysozyme (4-8mg/ml), and by incubating for 15-30 minutes to effect binding of the MBPto the particles before microscopic examination in a fluorescencemicroscope or counting in a cytofluorimeter (i.e., FAXSCAN, BectonDickinson Inc.) Cytofluorimetric counting was at a rate of about5,000-9,000 killed and denatured particles per second and requiringabout 2-30 seconds to count a total of 10,000-20,000 particles. Theuniformity of the dispersed cells following this sample treatmentprotocol is illustrated in the fluorescence photomicrograph presented inFIG. 2B, which was prepared from a normal urine sediment sample to whicha suspension of Candida albicans had been added. As illustrated in FIG.2B, most yeast forms were present either in single or budding doublecell forms, with only about one visible aggregate, clump or raft ofcells per 10 visual fields.

FIG. 2B shows a photomicrograph of fluorescence staining of Candidaalbicans grown for 48 hrs./37° C. on Sabouraud dextrose agar and thenadded to normal urine sediment, treated with alkaline treatments(supra), stained with F-NHS-labeled catalytically-disabled lysozymeprepared according to EXAMPLE 1, above (i.e., Preparation #1), andexamined as a wet mount using fluorescence microscopy. Most yeast formsstained were either single cell or budding double cell organisms withaggregates reduced to only one in every 10 microscopic visual fields.

For rapid cytometric analysis, this chemical sample pretreatmentprotocol was found to be crucial. In FIG. 2C, is shown a plot offluorescence intensity (x axis; FSC-H/FSC-Height) against particle size(y axis; SSC-H/SSC-Height) for a urine sediment collected from a normal(healthy) subject, (i.e., without bacteria or yeast particles), andsubject to the chemical treatment protocol disclosed above. The circlelabeled "YG" in FIGS. 2C and 2F indicates the region where fluorescentyeast particles, if present, would appear, i.e., referred to herein asthe "yeast gate". After cytometry counting, the data was subject tocomputerized selection and sorting process using Candida albicans as apositive control and normal human urine sediment particles as a negativecontrol. The selection process involved sorting the data for those datapoints having a particle size and a fluorescence intensity thatcorresponded visually (i.e., by screen display) with the size andfluorescence intensity for the positive yeast particle control whileexcluding the majority of the negative control urine sediment particles.In FIG. 2D, is shown an aliquot of the same urine sediment without thechemical treatment, i.e., urine sediment particles populate the yeastgate, and if yeast were present in the latter situation, they would notbe distinguishable from the urine residue particles based on size andfluorescence intensity, as shown from the data presented in FIG. 2E. InFIG. 2E, is shown another aliquot of the same urine sediment to which asuspension of Candida albicans was added. The sample was not subjectedto chemical treatment and the added yeast and urine particles havesimilar size distribution and both populate the `yeast gate`. In FIG.2F, is shown an aliquot of the same urine sample, with Candida albicansadded, and after the instant chemical treatment, i.e., yeast populatethe `yeast gate` and are separated from other urine sediment particles.

In FIGS. 2G-J is presented a plot of the selected yeast gate-data (yaxis; number of particles) for fluorescence intensity (x axis;FSC-H/FSC-Height) of killed and denatured Candida albicans particles.The experiment was designed to evaluate only the steps of alkalinetreatment and F-NHS-lysozyme staining, i.e., without N-acetylationand/or protease treatments. FIG. 2G shows the background in a controlsample from unstained and untreated yeast particles (i.e., the samplebackground without chemical treatment.) FIG. 2H depicts the control datacollected with unstained untreated aggregated yeast particles in buffer,rather than with urine sediment (i.e., the yeast background). FIG. 21depicts a plot of the fluorescence data obtained for an alkaline-treatedyeast urine sediment sample stained with F-NHS-lysozyme. TheF-NHS-lysozyme stained sample shows two relatively poorly resolved peaksof fluorescence staining, perhaps relating to the presence of doubletand singlet yeast forms in the sample. FIG. 2J depicts data obtained foran alkaline-treated yeast-buffer sample parallel to that of FIG. 2H,above, but stained with F-NHS-lysozyme. The two peaks of fluorescencecoincide with those in FIG. 2I indicating that the observed propertiesare independent of any possible urine/urea effects on the killed anddenatured yeast particles.

FIGS. 2K-2N present unsorted data showing the clear peak separationachieved between urine sediment particles and killed and denatured yeastparticles when alkaline treatment was followed by a step ofN-acetylation. In particular, data are shown from experiments in whichsuspensions of Candida albicans cells were added to 1 ml aliquots ofnormal urine. The aliquot samples were subjected to alkaline treatment(supra); followed by neutralization with 6 N HCl; and N-acetylation with5 μl (FIG. 2L) or 10 μl (FIG. 2N) of acetic anhydride at pH 8.0 in 400μl saturated bicarbonate buffer. The control results presented in FIG.2J and FIG. 2L show the yeast negative control background (unstained,i.e., "sample background", supra.) In FIGS. 2L and 2N are shown peaks ofyeast particles having fluorescence intensities, (i.e., "M1" channelpeak at about 1200-3000 FSC-H in FIGS. 2K and 2M), that are wellseparated from stained residual particles in the urine sediment (i.e.,at about 300-800 FSC-H.) The quantitative data collected in theseexperiments is summarized in TABLE 6.

                                      TABLE 6                                     __________________________________________________________________________    Cytofluorimetry Data: Quantitative Aspects                                    of the Data Presented in FIGS. 2K-2N                                                  Window.sup.a                                                                        Particles                                                                          %  Peak.sup.b                                                                        Peak Mean Median                                    Figure                                                                            Channel                                                                           L  R  Counted                                                                            Total                                                                            Counted                                                                           Channel 1                                                                          Intensity                                                                          Intensity                                 __________________________________________________________________________    2K  0   1.00                                                                             9646                                                                             10,000                                                                             100                                                                              383  17   38   15                                       2L  M1  254                                                                              9646                                                                               135                                                                              1.4                                                                               8  2654 1368 505                                       2M  0   1.00                                                                             9646                                                                             10,000                                                                             100                                                                              289  19   52   21                                       2N  M1  254                                                                              9646                                                                               245                                                                              2.5                                                                               18  274  958 392                                       __________________________________________________________________________     .sup.a Window, fluorescence intensity settings for lower and upper limits     .sup.b Peak Counted, number of particles counted at the peak of               fluorescence intensity.                                                  

To evaluate the combined advantages of alkaline treatment, acetylationand sorting data for particles conforming with a `yeast gate` (supra),Candida albicans was again used as a test fungi. Different numbers ofyeast cells, in suspension, were added to aliquots samples of normalhuman urine, and the samples subject to sedimentation and alkaline andacetylation treatments (supra.) Urine samples were stained by reactingfor 10, 15, 20 and 30 minutes at 37° C. with 0.01-1 mg/ml ofF-NHS-catalytically disabled lysozyme (Preparation #1, above) or ofF-NHS-lysozyme (i.e., wild-type enzyme conjugate, supra) and thenaliquots were subject to analysis by flow cytometric analysis in afluorescence cytometer (Beckton Dickinson Model LSYS II) at a flow rateof 1.0 μl/second and sufficient particles were commonly present so thatcounting could be concluded in about 3-4 seconds. After counting, asoftware-controlled gate setting was applied to the data and used tovisually select the `yeast gate` from a plot of particle size (i.e.,SSC) vs. fluorescence intensity. The resultant window excluded most ofthe fluorescence attributable to urine residue particles as well asbacteria. The results presented in TABLE 7, below, show the effects ofthe visual gate selection procedure on the particles counted and peakflourescence of control samples of urine spiked with Candida utilis(i.e., having small bacterial sized cells), Micrococcus lesodeikticus,and urine samples from three patients with UTI of unknown etiology andone with a bacterial UTI. All urine sediment samples were subjected tojust alkaline treatment followed by N-acetylation (supra.)

                  TABLE 7                                                         ______________________________________                                        Effect of Size Gating on Counting of Low Signal                               Fluorescence Particles in Urine Sediment Samples                              Sample              Yeast   Particles                                                                             Peak                                      No.    Sample       Gated   Counted Fluorescence                              ______________________________________                                        1      Urine sediment +                                                                           NO      10,000  300                                              C. utilis    YES        83    3                                        2      Urine sediment +                                                                           NO      10,000  379                                              M. lesodeikticus                                                                           YES        19    2                                        3      Patient A, UTI                                                                             NO      10,000  294                                              Bacterial    YES       522    17                                       4      Patient B, UTI                                                                             NO      10,000  278                                              Unknown      YES      8,670  277                                       5      Patient C, UTI                                                                             NO      10,000  200                                              Unknown      YES      1,614   47                                       6      Patient D, UTI                                                                             NO      10,000  223                                              Unknown      YES      5,185  221                                       ______________________________________                                    

The results show that 99% of all small bacteria-sized particles, i.e.,C. utilis and M. lesodeikticus, are removed from counting by thecomparative method of visually selecting a `yeast gate` withF-NHS-lysozyme stained Candida albicans positive control sample. Theresults also seem to confirm the known bacterial UTI infection inPatient #A, i.e., all of the particles counted fall outside of the rangeappropriate for yeast particles; and, in favor yeast infections inpatients #B and #C, since 87% and 52%, respectively, of the particlescounted fell within the range appropriate for yeast particles. Patient#C, may represent primarily a bacterial infection and/or a yeastsecondary infection since 16% particles counted fall within the regionselected for yeast.

In control experiments designed to investigate the sensitivity of theassay, known numbers of Candida albicans cells were added to normalurine sediment samples and the results were calculated and expressedgraphically as either the total number of YG particles counted permilliliter of urine (FIG. 3A), or the number of YG particles counted persecond (FIG. 3B.) The results presented in FIG. 3A show low-endsensitivity of the assay while those presented in FIG. 3B show high-endlinearity for signal production.

FIG. 3A depicts graphically the results of an experiment in whichdiffering volumes of a Candida albicans suspension (cells/ml) were addedto a normal human urine sediment, (i.e., 10 ml normal urine centrifuged,treated under just alkaline conditions and with protease treatment(supra.) The urine sediment was resuspended in 500 μl=20-foldconcentrated sample, and yeast cells were added to achieve yeastconcentrations in the range of about 1000-5000 cells/ml of urine. Fungiin the sample were stained by incubating for 30 minutes withF-NHS-lysozyme at a final concentration of 80 μg/ml. The number of YGparticles was quantified by cytofluorimetry using forward light scatterand flow cytometric counting. The x-axis expresses the microliters (μl)of yeast suspension added to the urine sediment and the y-axis thenumber of fluorescent stained YG particles detected per milliliter ofthe urine sedimented. (Curve fitting algorithm agreement, R2, was0.994.)

A scatter plot representing the size of particles (y axis; SSC-H countedat each fluorescence intensity (x-axis; FFC-H) in FIG. 3A showed thatthe majority of the particles counted in the treated and F-NHS-MBPstained urine sediment sample (according to the methods described inregard to FIG. 3A, supra) were either single cell or double-cell yeastparticles. However, the data also showed that a few aggregates stillexisted in alkaline and protease treated samples, accounting for a fewparticle counts lying outside the main YG fluorescence cluster.

FIG. 3B depicts graphically the results of an experiment in whichdiffering volumes of Candida albicans suspension (cells/ml) were addedto 10 ml of normal human urine. Following centrifugation the urine wasresuspended in 500 μl (urine sediment sample), and subject topreparative treatments, staining and cytofluorimetric analysis asdescribed in regards to FIG. 3A, above. The yeast were added to thewhole urine to achieve yeast concentrations in the range of about5,000-20,000 cells/ml urine, in this case, corresponding to 100-400cells/second in the urine sediment sample at a cytometer flow rate of1.01/second.

The results presented in FIG. 3B show a linear second order polynomialrelationship between the number of particles counted per second and thenumber of microliters of yeast input (cells/ml) into the assay over therange of about 1,000 to 5,000 cells/ml urine; and, without evidence thatthe assay was compromised in any way at the higher yeast inputs. Theresults presented in FIG. 3A show low-end assay sensitivity in the rangeof about 100 YG particles per milliliter of a urine sample, i.e., 1000particles total in a 10 ml urine sample. Additional experiments wereconducted in an attempt to determine the high-end limits of the assay(FIGS. 4 and 5), and to confirm linearity of signal generation at lowerinput analyte concentrations (FIG. 5) and in these experiments data areexpressed as YG particles counted per second at a cytometer flow rate of1 μl/sec. unless otherwise indicated.

FIG. 4 depicts graphically the results of an experiment in whichdiffering volumes of Candida albicans suspension (cells/ml) were addedto 10 ml of normal urine. Following centrifugation the urine wasresuspended in 500 μl (urine sediment sample), and subjected topreparative treatments, staining and cytofluorimetric analysis asdescribed above in regard to FIG. 3A. The yeast were added to the wholeurine to achieve yeast concentrations in the range of about50,000-200,000 cells/ml urine, i.e., 1,000-4,000 cells/second in theurine sediment sample at a cytometer flow rate of 1.0 μl/second. (Curvefitting algorithm agreement, R2, was 0.999.)

FIG. 5 depicts graphically the results of an experiment in whichdiffering volumes of Candida albicans suspension (cells/ml) were addedto 10 ml of normal urine. Following centrifugation the urine wasresuspended in 500 μl (urine sediment sample), and subject topreparative treatments, staining and cytofluorimetric analysis as indescribed in regards to FIG. 3A, above. The yeast were added to thewhole urine to achieve yeast concentrations in the range of about5,000-35,000 cells/ml urine, i.e., 100-700 cells/second in the urinesediment at a flow rate of 1.0 μl/second. (Curve fitting algorithmagreement, R2, was 1.0.)

FIG. 6 depicts graphically the results of an experiment in whichdiffering volumes of Candida albicans suspension (cells/ml) were addedto 10 ml of normal urine. Following centrifugation the urine wasresuspended in 500 μl (urine sediment sample), and subject topreparative treatments, staining and cytofluorimetric analysis asdescribed in regards to FIG. 3A, above. The yeast were added to thewhole urine to achieve yeast concentrations in the range of about50,000-200,000 cells/ml urine, i.e., 1,000-4,000 cells/second in theurine sediment at a flow rate of 1.0 μl/second. (Curve fitting algorithmagreement, R2, was 0.977.)

The combined results presented in FIGS. 3A, 3B, 4, 5 and 6 indicate anear linear relationship between signal generated and amount of analyteinput (i.e., yeast cells) into the assay with a dynamic range for theassay over about 2.3-logs of analyte concentration (i.e., 500-150,000cell/ml in urine), but with some fall-off in performance at 200,000cells/ml. The results show a low-end sensitivity of about 50-100cells/ml in urine (i.e., 5000 cells in a 10 ml urine sample,concentrated to 500 μl in a urine sediment, giving about 10particles/μl/ second through the cytometry detector.) The lower limitfor linearity of detection, quantification, in this particular assay wasin the range of about 1,500 to about 3,000 cells/ml of a urine sample: anumber that may correspond to about 500-1000 cells. An acceptablesignal-noise ratio was achieved through the alkaline and N-acetylationsample treatments, and use of an F-NHS-MBP yeast positive control toselect a yeast gate setting for sorting and accepting data points.

To test stability of the killed and denatured disaggregated and stainedparticles (i.e., just alkaline and/or protease treated and stained)prepared and F-NHS-MBP stained urine sediment sample, samples stainedwith F-NHS-lysozyme according to FIGS. 2 and 3A, supra, were stored at4° C. for 3 days and then assayed by flow cytometry. The resultspresented in FIG. 7 show that the signal generation by the sample wasstable to storage at 4° C.

FIG. 7 depicts graphically the results of an experiment in whichdiffering volumes of a Candida albicans suspension (cells/ml) were addedto a normal urine sediment, (i.e., 10 ml normal urine centrifuged,treated under alkaline conditions and with protease-supra, and thenresuspended in 500 μl=20-fold concentrated sample), to achieve yeastconcentrations in the range of about 2,500-20,000 cells/ml of urine.Yeast were stained with F-NHS-labeled MBP, as described in FIG. 3A, andthen stored at 4° C. for 3 days prior to analysis. The number of fungalparticles was quantified by cytofluorimetry using forward light scatterand flow cytometric counting. The x-axis expresses the microliters ofyeast suspension added to the urine sediment and the y-axis the numberof fluorescent stained particles detected per milliliter of the urinesediment. (Curve fitting algorithm, R2, was 0.994.)

Additional experiments were conducted to verify that the alkalinetreatment conditions were effective for treatment of clinical samplescontaining differing numbers of cells; that the assay was functionalwithin the range of analyte (yeast cells/ml) concentrations encounteredin clinical practice; and that linearity was preserved when usingclinical samples. For these experiments a urine sample was collectedfrom patient #1201 with UTI confirmed by culture. The biological samplewas subject to treatment as follows: namely, 1, 2, 3, 4, 5, or 9 ml ofurine were centrifuged in a clinical centrifuge (3000 rpm/10 minutes at4° C.) to prepare a urine sediment. Each respective urine sediment wasthen treated under alkaline conditions only, i.e., 500 μl/1 N NaOH, and,since some visual aggregates still remained the sample was next treatedwith 0.25% trypsin for 30 minutes (supra) to disaggregate remainingurine sediment particles. The respective killed and denatured urineresidue sediments were resuspended in 500 μl of PBS and then stainedwith F-NHS-labeled catalytically disabled-lysozyme (Preparation #1,Method 1.) The respective sediments were washed with PBS andresuspending in 500 μl PBS for flow fluorimetry, and stained for 30minutes at 37° C. with 80μg/ml of the catalytically inactiveF-NHS-lysozyme. Cytometric methods were as described in regard to FIG.3A, above, with sampling was at 60 μl/minute using the undiluted treated500 μl urine sediment sample (supra.) Sample counting was complete(i.e., 97-99% confidence interval) at 4 seconds for the 1 ml urinesample (i.e., 5,400 fungal cells/sec.) and at 3 seconds for the 2-9 mlurine samples (i.e., 6,500 cells/sec.-8,800 cells/sec.) The resultspresented in FIG. 8 show that patient #1201 had about 5,400-8,800cells/μl/second in the respective different urine sediments(corresponding to 54,000-2,700,000 yeast/ml in urine), i.e., at the highoperating range of the assay. The slope of the curve in FIG. 8, as wellas, the y-intercept value, each defining a parameter for the severity ofthe UTI infection in patient #1201. The results presented in FIG. 8 showthat sufficient particles are present in certain clinical samples toallow for collection of only small urine volumes and/or dilution ofurine sediment samples prior to analysis. Thus, titration curves may beconstructed using differing amounts of urine samples (e.g., as in FIG.7), or alternatively, by preparing a dilution series from a freshlyprepared and stained (or 4° C. stored) urine sediment. The dilutionseries can be constructed to bring the number of particles counted/μl ofurine sediment sample/second within the range of about 1500-3000. Thedata collected in the assay may be plotted as cell counts per microliterurine sediment per second versus the dilution of urine sediment input tothe assay, (i.e., as in FIG. 8), and the plotted data defines a slope ofa curve with a y-intercept. Both the value for the slope and for they-intercept relate to the severity of an infection in the patient fromwhich the urine sample was obtained. Quantitative aspects of the dataobtained in this experiment are summarized in TABLE 8, below.

                  TABLE 8                                                         ______________________________________                                        Sample  MBP-     Particles                                                                              Mean Fluorescence                                                                        YG                                       Volume (ml)                                                                           conjugate*                                                                             Counted  Intensity  Particles/sec.                           ______________________________________                                        1       +        20,000   515        5,400                                    2       +        20,000   828        6,500                                    3       +        20,000   813        7,000                                    4       +        20,000   1199       7,000                                    5       +        20,000   1586       7,200                                    9       +        20,000   1854       8,800                                    Negative                                                                              -        20,000    6         --                                       Control                                                                       ______________________________________                                         *MBP-conjugate, murein binding polypeptideconjugate, i.e., catalytically      inactive FNHS-lysozyme; 500 μl total final volume; window setting for      fluorescence intensity L, 1.00 and R 9646.                               

The results show that the assay and alkaline treatment method, arecapable of quantifying fungal particles in a killed and denatured urinesediment residue in the absence of a step of N-acetylation.N-acetylation, however, is effect to increase the signal strength in theassay, improve low end sensitivity and reproducibility.

The results presented in FIGS. 9A, 9B, 10A, 10B and 11A-D showperformance of the assay with clinical urine samples collected frompatients with urinary tract infections. All samples were subject tosedimentation (3000 rpm in a clinical centrifuge, supra); alkaline-(2NNaOH/30 minutes/60° C., supra) followed by neutralization-(6N HCl,supra), and acetylation-(10 μl acetic anhydride, pH 8 bicarbonatebuffer, supra) treatments; staining with F-NHS-lysozyme (80 μg/ml,supra); and, fluorocytometric particle counting (1 μl/sec.) as describedin regard to FIG. 3A, above (i.e., without protease treatment).Fluorescence data points in FIGS. 9A, 10A, 11A and 11C were ungatedwhile those presented in FIGS. 9B, 10B, 11B and 11D were sorted andselected for YG particles falling with a `yeast gate` (supra.) (Thenumber of YG particles counted during sample analysis in regards toFIGS. 11C and 11D, i.e., 1800 YG particles/sec., allowed shortercounting times.)

The data presented in FIGS. 9A and 9B was recorded after analysis of aurine sample from a patient having a confirmed (by culture) bacterialUTI and most killed and denatured particles were clustered into a singlepeak having a fluorescence intensity of about 107 (i.e., FSC-H.)Analysis of the data collected in the instant assay supports diagnosisof a bacterial urinary tract infection. Interestingly, after sorting thedata to remove most urinary sediment particles and bacterial particles(i.e., the peak in FIG. 9A), a small residual broad peak of YG particleswas observed with a mean fluorescence intensity at about 1892 FSC-H. Itis theoretically possible that this patient had developed a low-levelsecondary yeast infection.

The data presented in FIGS. 10A, 10B, was recorded after analysis of aurine sample from a patient with a confirmed yeast UTI, and similarly,and data presented in FIGS. 11A, 11B, 11C and 11D was recorded afteranalysis of urine sediment samples collected one day apart (i.e., FIGS.11A-B, day 1; FIG. 11C-D, day 2) from a patient with a UTI of unknownetiology. The results presented in FIGS. 10A-B and 11A-D show YGparticles in the samples, and support a diagnosis of a yeast UTI.Importantly, the fluorescence intensity data presented in FIGS. 11C-Dwas recorded over a much shorter sampling time (i.e., at a counting rateof 1800 cells/ second) than that recorded in FIGS. 11A-B and the totalYG particles counted in FIGS. 11C-D were nearly three times greater thanthose recorded in FIGS. 11A-B. The results presented in FIGS. 11A-Dwould suggest that if anti-bacterial therapy was administered to thisparticular patient, it was not effective in eliminating the yeastinfection.

Materials

Protein assay reagents, ion exchange resins (e.g., BioRex-70), and gelfiltration resins were from Bio-Rad Laboratories (Richmond, Calif.).Chitin, chitosan, buffer salts, and electrophoresis standards were fromSigma Chemical Co. (St. Louis, Mo.). Acrylamide: bisacrylamide solution(37.5:1) was from Amresco (Solon, Ohio).

Sepharose 4B and Sephadex 4B, 6B and Sephadex G75 were from PharmaciaFine Chemicals (Uppsala, Sweden.) Calibration beads having a diameter of0.5μ were from Becton-Dickinson.

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While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

We claim:
 1. A murein binding polypeptide in vitro diagnostic reagentfor detecting a eubacteria or a fungus in a biological sample,whereinsaid murein binding polypeptide in vitro diagnostic reagentcomprises a murein binding polypeptide chemically conjugated to a signalgenerating compound; said murein binding polypeptide comprises acatalytically inactive enzyme polypeptide having a binding site capableof binding to either a eubacterial murein compound or a fungal mureinlike compound with a binding affinity of about 5×10⁻⁷ L/mol to about5×10⁻⁹ L/mol; and wherein said conjugate comprising said murein bindingpolypeptide and signal generating compound is effective when bound tosaid eubacteria or said fungus to produce a detectable signal in adiagnostic assay format.
 2. The murein binding polypeptide in vitrodiagnostic reagent of claim 1, wherein said signal generating compoundis selected from the group consisting of an enzyme, a fluorophore, aphycobilin, a biotin, an avidin, a streptavidin, a bioluminescentcompound, a chemiluminescent compound, a histochemical dye, and aradiolabeled compound.
 3. The murein binding polypeptide in vitrodiagnostic reagent of claim 1, wherein said catalytically inactiveenzyme polypeptide comprises a derivative of a wild-type enzyme selectedfrom the group consisting of a mutant enzyme, a recombinant enzyme, anda chemically inactivated enzyme.
 4. The murein binding polypeptide invitro diagnostic reagent of claim 3, wherein said wild-type enzymepolypeptide is selected from the group consisting of anacetyl-muramoyl-D,L-Alanyl amidase, a bacterial cell wall penicillinbinding protein, an alanyl D,D- or D,L-endopeptidase, a D,D- orD,L-carboxypeptidase, a transglycosyl transferase, a peptidyltransferase, a muramoyl isomerase, a muramoyl transglycosylase, a mureinautolysin, a murein hydrolase, and a lysozyme.
 5. The murein bindingpolypeptide in vitro diagnostic reagent of claim 1, further comprisingan additive selected from the group consisting of a stabilizer, abuffer, an emulsifier and an agent for promoting a binding interactionbetween said murein binding polypeptide and said murein or said mureinlike compound.
 6. The murein binding polypeptide in vitro diagnosticreagent of claim 1, wherein said catalytically inactive enzymepolypeptide comprises a portion of a wild-type enzyme having saidbinding site.
 7. A murein binding polypeptide in vitro diagnosticreagent for detecting a eubacteria or a fungus in a biological sample,whereinsaid murein binding polypeptide in vitro diagnostic reagentcomprises a multimeric murein binding polypeptide chemically conjugatedto a signal generating compound; said multimeric murein bindingpolypeptide comprises two or more murein binding polypeptides eachindependently comprising a catalytically inactive enzyme polypeptidehaving a binding site capable of binding to either a eubacterial mureincompound or a fungal murein like compound with a binding affinity ofabout 5×10⁻⁷ L/mol to about 5×10⁻⁹ L/mol; wherein said conjugatecomprising said murein binding polypeptide and signal generatingcompound is effective when bound to said eubacteria or said fungus toproduce a detectable signal in a diagnostic assay format.
 8. The mureinbinding polypeptide in vitro diagnostic reagent of claim 7, wherein saidtwo or more catalytically inactive enzyme polypeptides each comprise aderivative of a wild-type enzyme selected from the group consisting of amutant enzyme, a recombinant enzyme, and a chemically inactivatedenzyme.
 9. The murein binding polypeptide in vitro diagnostic reagent ofclaim 8, wherein said two or more murein binding polypeptides comprisederivatives of different wild-type enzymes.
 10. The murein bindingpolypeptide in vitro diagnostic reagent of claim 9, wherein said two ormore different catalytically inactive enzymes comprises a chimericpolypeptide.